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WO2025241209A1 - Mutant de protéine f de pré-fusion de vrs, sa préparation et son utilisation - Google Patents

Mutant de protéine f de pré-fusion de vrs, sa préparation et son utilisation

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Publication number
WO2025241209A1
WO2025241209A1 PCT/CN2024/096123 CN2024096123W WO2025241209A1 WO 2025241209 A1 WO2025241209 A1 WO 2025241209A1 CN 2024096123 W CN2024096123 W CN 2024096123W WO 2025241209 A1 WO2025241209 A1 WO 2025241209A1
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Prior art keywords
mutant
fusion
rsv
protein
seq
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/CN2024/096123
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English (en)
Chinese (zh)
Inventor
崔保峰
胡浩
蒋浩然
滕忠坤
周茹
王姗姗
王文宇
王婧
周翠云
郑海东
于旭博
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Juwei Biotech Co Ltd
Original Assignee
Suzhou Juwei Biotech Co Ltd
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Filing date
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Publication of WO2025241209A1 publication Critical patent/WO2025241209A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • 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
    • 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
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • This application belongs to the field of biotechnology and relates to a mutant of the RSV fusion pre-fusion F protein, its preparation and application.
  • Respiratory syncytial virus is a highly contagious respiratory pathogen and a leading cause of hospitalization and death in infants under 6 months of age worldwide. It can cause severe lower respiratory tract infections in infants, the elderly, and immunocompromised individuals. WHO studies on the etiology of acute respiratory infections in children show that RSV accounts for more than 60% of all respiratory illnesses in children. In 2019, there were 33 million cases of acute lower respiratory tract infection (ALRI) caused by RSV in children aged 0-5 years globally, resulting in 3.6 million hospitalizations. Of these hospitalized children, 101,000 ultimately died, with infants aged 0-6 months accounting for approximately 46% of the deaths. This places a significant economic burden on public health resources worldwide.
  • ARI acute lower respiratory tract infection
  • RSV belongs to the Paramyxoviridae family. Its genome consists of 15,200 nucleotides and contains 10 genes encoding 11 proteins. Among these structural proteins are three transmembrane surface glycoproteins: glycoprotein G, fusion protein F, and small hydrophobic protein SH. Fusion protein F and glycoprotein G are key targets for neutralizing antibodies. However, the highly variable conformational changes of glycosylated glycoprotein G and fusion protein F pose challenges to vaccine development.
  • Fusion protein F is a type I membrane fusion protein.
  • the virus-encoded F0 protein forms a trimer in the endoplasmic reticulum and contains two furin cleavage sites. After furin cleavage, it forms two proteins, F1 and F2, and a Pep27 polypeptide fragment.
  • the F1 protein contains its N-terminal hydrophobic fusion peptide and two heptapeptide repeat regions (HRA and HRB).
  • HRA and HRB heptapeptide repeat regions
  • mutants containing DS-Cav1 S155C, S290C, S190F, V207L described in CN105473604B, US10017543B2, and US11130785B2; and mutants containing SC-D...
  • Stable pre-F mutants of M and SC-TM (N67I, S215P, E487Q); pre-F mutants described in US20210023200A1 and CN108738312A (S55C, L188C, L142C, N371C); and pre-F mutants containing mutations at the L141C, L142C, and L373C positions described in CN114929877A.
  • Traditional mutational strategies mainly include disulfide bond mutations, cavity filling, or charge balancing, aiming to maintain a stable conformation of pre-F or increase pre-F expression levels.
  • the stabilization of pre-type F proteins remains insufficient, and structure-based optimization is under ongoing investigation.
  • the main objective of this application is to provide a mutant of the F protein before RSV fusion, its preparation, and its application.
  • the technical solution includes:
  • a mutant of the F protein before RSV fusion wherein the ⁇ 3 helical region of the F1 polypeptide has a cysteine substituent 1 and the ⁇ 3 sheet region has a cysteine substituent 2, wherein the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond.
  • the species source of the RSV pre-fusion F protein is human or bovine.
  • the F1 polypeptide of the RSV fusion pre-fusion F protein has the amino acid sequence shown in SEQ ID NO. 51 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 51.
  • the F1 polypeptide of the pre-fusion F protein of RSV has the amino acid sequence shown in SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO. 75. In some embodiments of this application, the F1 polypeptide of the pre-fusion F protein of RSV has the amino acid sequence shown in SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO. 75. Peptides satisfy one or more of the following conditions:
  • the F1 polypeptide of the RSV fusion pre-fusion F protein has one or more of the following mutations: I379V and M447V.
  • the F1 polypeptide of the RSV fusion pre-fusion F protein has the amino acid sequence shown in SEQ ID NO. 52.
  • the distance between the cysteine substituent 1 and the cysteine substituent 2 is...
  • the ⁇ 3 helical region of the F1 polypeptide of the mutant has one or more of the following mutations: E163C, K166C, I167C, A170C, and L171C.
  • the ⁇ 3 sheet region of the F1 polypeptide of the mutant has one or more of the following mutations: A177C, V179C, and L181C.
  • the F1 polypeptide of the mutant has one of the following combinations of mutations:
  • Group 1 consists of E163C and L181C.
  • Group 2 consists of K166C and V179C.
  • Group 3 consists of I167C and V179C.
  • Group 4 consists of A170C and V179C.
  • Group 5 consists of A170C and A177C, and,
  • Group 6 consists of L171C and A177C.
  • the mutant also has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C.
  • the mutant has one set of the following combinations of mutation sites:
  • the mutant does not contain a furin cleavage site fragment.
  • the mutant does not contain the pep27 peptide, and the C-terminus of the F2 peptide and the N-terminus of the F1 peptide are directly linked by an amide bond or indirectly linked by a flexible short peptide.
  • the flexible short peptide may optionally be GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPG, GSGS, GGGS, GPGS, GGGG, GGSG, GGSG, SGGG, GSSG, SGSG, GGPGG, or GGGGS.
  • the C-terminus of the F1 polypeptide of the mutant is linked to a tag fragment and/or an aggregation motif.
  • the tag fragment includes a 6His polypeptide.
  • the aggregation motif is as shown in SEQ ID NO.48.
  • the mutant F2 polypeptide has the amino acid sequence shown in SEQ ID NO.49 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO.49.
  • the mutant F2 polypeptide has the amino acid sequence shown in SEQ ID NO.49, SEQ ID NO.76 or SEQ ID NO.77.
  • the F2 polypeptide of the mutant satisfies one or more of the following conditions:
  • the C-terminus does not contain NN, and,
  • the F2 polypeptide of the mutant has the amino acid sequence shown in SEQ ID NO.50.
  • the amino acid sequence of the mutant is shown as any one of SEQ ID NO.12 to SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.42 to SEQ ID NO.47 and SEQ ID NO.53 to SEQ ID NO.73.
  • nucleic acid molecule that encodes a mutation of the RSV pre-fusion F protein described in the first aspect. Variants.
  • a carrier that includes the nucleic acid molecule described in the second aspect.
  • an engineered cell that expresses a mutant of the RSV pre-fusion F protein as described in the first aspect, or that includes the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect.
  • a method for producing a mutant of the RSV fusion-pre-fusion F protein comprising the following steps:
  • the engineered cells described in the fourth aspect are cultured, and the mutant of the RSV fusion-pre-fusion F protein is isolated from the resulting culture supernatant.
  • an immune composition comprising a mutant of the RSV pre-fusion F protein as described in the first aspect or a nucleic acid molecule as described in the second aspect, and an immune adjuvant.
  • the immune adjuvant includes one or more of aluminum salt adjuvants, surfactants, polynucleotides, lipopolysaccharides, liposomes, and oil emulsion adjuvants.
  • a mutant of the RSV pre-fusion F protein described in the first aspect is provided for use in the preparation of a respiratory syncytial virus antibody detection kit.
  • a respiratory syncytial virus antibody detection kit which includes a mutant of the RSV pre-fusion F protein described in the first aspect.
  • a method for preventing and treating lower respiratory tract infections caused by respiratory syncytial virus comprising the following steps: administering a therapeutically effective amount of the immune composition described in the sixth aspect to a subject.
  • a method for detecting or separating RSVF-binding antibodies in a sample comprising the following steps:
  • the mutant of the RSV pre-fusion F protein described in the first aspect is contacted with an RSVF-binding antibody in the sample to form an immune complex;
  • the immune complex is detected to detect or isolate RSV F-binding antibodies in a sample.
  • Figure 1 shows a schematic diagram of the RSV F0 protein structure (A, B, bovine);
  • Figure 2 is a schematic diagram of the structure of the constructed stable mutant RSV F protein polypeptide monomer
  • Figure 3 is a schematic diagram of the structure of the constructed stable mutant RSV F protein polypeptide trimer
  • Figure 4 is a diagram showing the presence of 7 pairs of disulfide bonds in the natural RSV F0 precursor protein or mature F protein.
  • Figure 5 shows a schematic diagram of the three-dimensional structure of the RSV F protein monomer (left) and a magnified schematic diagram of the local area located in the ⁇ 3 and ⁇ 3 sheets (right);
  • Figure 6 shows the amino acids involved in the ⁇ 3 helix and ⁇ 3 fold positions in this application, as well as the interatomic distances between two amino acids that readily form disulfide bonds.
  • Figure 7 shows the SDS-PAGE analysis results of the mutant under reducing conditions; where JW-05 is the mutant monomer and JW-05-T4 is the mutant trimer.
  • Figure 8 shows the high-performance liquid chromatography (HPLC) analysis results of the RSV pre-F mutant; Figure 8a shows the mutant monomer, and Figure 8b shows the mutant trimer.
  • HPLC high-performance liquid chromatography
  • Figure 9 shows the expression statistics of RSV pre-F monomers formed by the ⁇ 3/ ⁇ 3 position mutation
  • Figure 10 shows the thermal stability statistics of RSV pre-F monomers formed by the ⁇ 3/ ⁇ 3 positional mutation under 50°C treatment
  • Figure 11 shows the statistical graph of RSV pre-F mutant trimer expression levels
  • Figure 12 shows the thermal stability statistics of RSV pre-F mutant trimer under 50°C treatment
  • FIG. 13 shows the RSV pre-F mutant trimer and D25 monoclonal antibody ( The binding specificity of epitopes;
  • FIG 14 shows the RSV pre-F mutant trimer and AM22 monoclonal antibody ( The binding specificity of epitopes
  • Figure 15 shows the binding specificity of RSV pre-F mutant trimer to AM14 monoclonal antibody (trimeric epitope);
  • Figure 16 shows the results of antigen-specific antibody level detection for RSV pre-F mutants
  • Figure 17 shows the results of neutralizing antibody detection in RSV pre-F mutant
  • Figure 18 shows the adsorption rate of neutralizing antibodies in serum by RSV pre-F mutant.
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR”), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").
  • “one or more” means one or more than or equal to two.
  • suitable refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.
  • first aspect “second aspect,” “third aspect,” “fourth aspect,” etc.
  • second aspect “third aspect”
  • fourth aspect is used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features.
  • first,” “second,” “third,” “fourth,” etc. serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.
  • the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C, or ⁇ 1°C.
  • % (w/w) and wt% both represent weight percentage
  • % (v/v) refers to volume percentage
  • % (w/v) refers to mass-volume percentage
  • This application provides a method for stabilizing RSV F0 in a pre-F state through amino acid mutation, and discloses a disulfide bond mutation position not addressed in existing technology or patents.
  • This position involves introducing a disulfide bond mutation between ⁇ 3 and ⁇ 3 of the RSV F protein, and combinations of mutations based on this mutation can interact with recognition...
  • the site-specific monoclonal antibody binds and has good stability.
  • the mutant multimer constructed from this not only significantly increases the expression level of pre-F protein, but also has good immunogenicity and can induce high levels of neutralizing antibodies, showing great potential for vaccine development.
  • this application discovers another type of disulfide bond mutation site, primarily between ⁇ 3 and ⁇ 3, which is not addressed in existing technologies or patents. Individual disulfide bond mutations at this site, or combinations of mutations at other sites, can be identified...
  • the site-specific monoclonal antibody binds at high levels and exhibits good stability, demonstrating great potential for developing RSV vaccines.
  • the embodiments of this application include:
  • an embodiment of this application provides a mutant of the F protein before RSV fusion, wherein the ⁇ 3 helical region of the F1 polypeptide has a cysteine substituent 1 and the ⁇ 3 sheet region has a cysteine substituent 2, wherein the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond.
  • amino acid substitution One amino acid in an antigen is replaced by another amino acid, or an amino acid is missing.
  • an amino acid in an antigen may be replaced by an amino acid from a homologous protein.
  • Homologous proteins Proteins that have similar structures and functions, for example, proteins from two or more species or viral strains that have similar structures and functions.
  • the RSVF protein from RSVA is homologous to the RSVF protein from bovine RSV.
  • Homologous proteins share similar protein folding features and can be considered structural homologs.
  • Homologous proteins typically share a high degree of sequence conservation, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence conservation, and a high degree of sequence identity, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • RSV RSV
  • human subtype A human subtype B
  • bovine subtype Within each RSV subtype, there are individual strains for each subtype.
  • the amino acid sequence of the remaining fragments can be the wild-type sequence, or it can be a sequence with other mutations on the basis of the wild-type sequence through artificial or natural means. The introduction of other mutations can still stabilize the mutant.
  • the F1 polypeptide of the pre-fusion F protein of the RSV has the amino acid sequence shown in SEQ ID NO. 51 or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%) amino acid sequence with respect to the amino acid sequence shown in SEQ ID NO. 51. Homology of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% (%).
  • the F1 polypeptide of the pre-fusion F protein of the RSV has the amino acid sequence shown in SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO. 75.
  • the F1 polypeptide of the pre-RSV fusion F protein satisfies one or more of the following conditions:
  • the F1 polypeptide of the pre-fusion F protein of the RSV has one or more of the following mutations: I379V and M447V.
  • the F1 polypeptide of the pre-fusion F protein of the RSV has the amino acid sequence shown in SEQ ID NO. 52.
  • the distance between the cysteine substituent 1 and the cysteine substituent 2 is... (For example, 2, 3, 4, 5, 6, 7, ).
  • the ⁇ 3 helical region of the F1 polypeptide of the mutant has one or more of the following mutations: E163C, K166C, I167C, A170C, and L171C.
  • the ⁇ 3 sheet region of the F1 polypeptide of the mutant has one or more of the following mutations: A177C, V179C, and L181C.
  • the F1 polypeptide of the mutant has one of the following combinations of mutations:
  • Group 1 consists of E163C and L181C.
  • Group 2 consists of K166C and V179C.
  • Group 3 consists of I167C and V179C.
  • Group 4 consists of A170C and V179C.
  • Group 5 consists of A170C and A177C, and,
  • Group 6 consists of L171C and A177C.
  • the mutant further has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C.
  • S55C is located in the F2 polypeptide.
  • S155C, S180C, S186C, L188C, S190F, V207L, and S290C are located in the F1 polypeptide.
  • the mutant has one set of the following combinations of mutation sites:
  • the mutant does not contain a furin cleavage site fragment.
  • the mutant does not contain the pep27 peptide, and the C-terminus of the F2 peptide and the N-terminus of the F1 peptide are directly linked by an amide bond or indirectly linked by a flexible short peptide.
  • linking may refer to the creation of a continuous molecule from two molecules; for example, linking two other polypeptides into a continuous polypeptide, or covalently linking a carrier molecule or other molecule to an immunogenic polypeptide, such as the mutants disclosed herein. Linking may be carried out chemically or through recombination.
  • the flexible short peptide may optionally be GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPG, GSGS, GGGS, GPGS, GGGG, GGSG, GGSG, SGGG, GSSG, SGSG, GGPGG, or GGGGS.
  • the C-terminus of the F1 polypeptide of the mutant is linked to a tag fragment and/or an aggregation motif.
  • the tag fragment includes a 6His polypeptide.
  • the aggregation motif is as shown in SEQ ID NO.48.
  • the mutant F2 polypeptide has the amino acid sequence shown in SEQ ID NO. 49 or shares at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the amino acid sequence shown in SEQ ID NO. 49.
  • the mutant F2 polypeptide has the amino acid sequence shown in SEQ ID NO. 49, SEQ ID NO. 76, or SEQ ID NO. 77.
  • the mutant F2 polypeptide satisfies one or more of the following conditions: 1) it does not contain NN at the C-terminus, and 2) it has the following mutation: P102A.
  • the mutant F2 polypeptide has the amino acid sequence shown in SEQ ID NO. 50.
  • the amino acid sequence of the mutant is shown as any one of SEQ ID NO.12 to SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.42 to SEQ ID NO.47 and SEQ ID NO.53 to SEQ ID NO.73.
  • the RSV fusion pre-fusion F protein mutant provided in this application has a stable conformation and can induce an immune response in the subject as an antigen/immunogen, producing antibodies.
  • the subject is an animal.
  • An animal is a living multicellular vertebrate or invertebrate, including, for example, mammals.
  • the term mammal includes human mammals and non-human mammals.
  • the term "subject" includes human and veterinary subjects, such as non-human primates. Therefore, administration to a subject can include administration to human subjects.
  • Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), livestock (e.g., cattle, horses, pigs, sheep, and goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).
  • Antibody A polypeptide, found in nature, encoded by one or more immunoglobulin genes or fragments thereof, that specifically binds to and recognizes an analyte (e.g., an antigen or immunogen), such as RSVF protein or an antigenic fragment thereof.
  • Immunoglobulin genes include ⁇ , ⁇ , ⁇ , ⁇ , 6, ⁇ , and ⁇ constant region genes, as well as numerous immunoglobulin variable region genes.
  • the term "antibody” as used herein includes, for example, modifications of a complete antibody and antibody fragments synthesized de novo using recombinant DNA methods.
  • Antibodies exist, for example, in the form of intact immunoglobulins and in the form of various well-characterized antibody fragments.
  • Fab, Fv, and single-chain Fv (SCFv) bound to RSVF proteins will be RSVF protein-specific binders.
  • scFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin is linked to the heavy chain variable region of an immunoglobulin by a linker, while in dsFv, the chains have been mutated to introduce disulfide bonds to stabilize chain association.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous binding antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd edition, W.H. Freeman & Co., New York, 1997.
  • Antibody fragments are defined as follows: (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule produced by digesting an intact antibody with the enzyme papain to obtain a portion of the intact light chain and a heavy chain; (2) Fab′, a fragment of an antibody molecule obtained by treating an intact antibody with pepsin and then reducing it to obtain a portion of the intact light chain and a heavy chain; each antibody molecule yields two Fab′ fragments; (3) (Fab′)2, a fragment of an antibody obtained by treating an intact antibody with the enzyme pepsin without subsequent reduction; (4) F(ab′)2, a dimer in which two Fab′ fragments are held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing variable regions of the light chain and variable regions of the heavy chain, represented by two chains; and (6) a single-chain antibody (“SCA”), a genetically engineered molecule in the form of a genetically fused single-chain molecule containing variable regions
  • immunoglobulins typically have heavy (H) chains and light (L) chains linked together by disulfide bonds.
  • Disclosed antibodies can be of different types.
  • Each heavy and light chain contains constant and variable regions (also referred to as “domains”).
  • the heavy and light chain variable domains combine to specifically bind the antigen.
  • only the heavy chain variable domain is required.
  • naturally occurring camel antibodies consisting only of the heavy chain are functional and stable in the absence of the light chain (see, for example, Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996).
  • the light and heavy chain variable domains contain “framework” regions interrupted by three hypervariable regions (also referred to as “complementarity-determining regions” or “CDRs”) (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991).
  • CDRs complementarity-determining regions
  • the sequences of the frame regions of different light or heavy chains are relatively conserved within a species.
  • the frame region of an antibody which is a combined frame region of light and heavy chains, is used to locate and align the CDR in three-dimensional space.
  • CDRs are primarily responsible for binding to epitopes of antigens.
  • the amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27: 55-77, 2003; “IMGT” numbering scheme).
  • the CDRs for each chain are typically referred to as CDR1, CDR2, and CDR3 (from N to C), and are usually defined by specific CDRs.
  • the CDRs are identified by the position of the chain. Therefore, VHCDR3 is located in the variable domain of the heavy chain of the antibody in which it is contained, while VLCDR1 is a CDR1 derived from the variable domain of the light chain of the antibody in which it is contained.
  • Light chain CDRs are sometimes referred to as CDRL1, CDRL2, and CDRL3.
  • Heavy chain CDRs are sometimes referred to as CDRH1, CDRH2, and CDRH3.
  • Antigen A compound, composition, or substance that can stimulate the production of an antibody or T-cell response in an animal, including compositions injected or absorbed into the animal.
  • An antigen is a product that reacts with a specific humoral or cellular immune response, including those induced by heterologous antigens, such as the disclosed mutant of the RSV pre-fusion F protein.
  • antigens include (but are not limited to) polypeptides, peptides, lipids, polysaccharides, combinations thereof (e.g., glycopeptides), and nucleic acids containing antigenic determinants, such as those recognized by immune cells.
  • antigens include peptides derived from the pathogen of interest, such as RSV.
  • antigens are derived from RSV, such as antigens comprising modified RSVF proteins stabilized in a pre-fusion conformation.
  • An “epitope” or “antigenic determinant” refers to an antigenic region that reacts with B and/or T cells.
  • Immunogen A protein or portion thereof capable of inducing an immune response in mammals, such as mammals infected with or at risk of infection with pathogens. Administration of an immunogen can result in protective and/or active immunity against the pathogen of interest. Examples include the PreF mutant provided in the embodiments of this application.
  • Immune response The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus.
  • the response is specific to a particular antigen (“antigen-specific response”).
  • the immune response is a T cell response, such as a CD4+ or CD8+ response.
  • the response is a B cell response and results in the production of specific antibodies.
  • the following six groups each contain amino acids that are conservedly substituted for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and [0182] 6) phenylalanine (F), tyrosine (Y), tryptophan (W).
  • Epitopes Antigenic determinants. These are specific chemical groups or peptide sequences on antigenic molecules that induce specific immune responses; for example, an epitope is an antigenic region that reacts with B and/or T cells. Antibodies bind to specific antigenic epitopes, such as epitopes of RSVF proteins, for example, the D25 or AM22 epitopes present in the pre-fusion conformation of the RSVF protein. Epitopes can be formed from consecutive or discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids generally remain exposed to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents.
  • Epitopes typically comprise at least 3 and more usually at least 5, about 9, or about 8-10 amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and nuclear magnetic resonance. Epitopes may also include post-translational modifications of amino acids, such as N-linked glycosylation.
  • a “target epitope” is a specific epitope on an antigen that specifically binds to an antibody of interest, such as a monoclonal antibody.
  • a target epitope includes amino acid residues that contact the antibody of interest so that the target epitope can be selected by determining the amino acid residues that contact the antibody of interest.
  • amino acids in peptides, polypeptides, or proteins are typically linked together chemically via amide bonds (CONH).
  • amino acids can be linked together by other chemical bonds.
  • Peptide Modification Peptides, for example, stabilized in their pre-fusion conformation.
  • the mutants in this application can be modified, for example by substitutions including amino acids relative to the native RSV protein sequence, or by various chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally other desired properties.
  • the carboxylic acid group of the protein whether carboxyl terminus or side chain, can be provided in the form of a pharmaceutically acceptable cationic salt or esterified to form a C1-C16 ester, or converted to an amide of formula NR1R2, wherein R1 and R2 are each independently H or C1-C16.
  • Alkyl groups, or combinations thereof, can form heterocycles, such as 5- or 6-membered rings.
  • the amino group of the peptide can be in the form of pharmaceutically acceptable acid addition salts such as HCl, HBr, acetates, benzoates, toluenesulfonates, maleates, tartrates, and other organic salts, or can be modified into C1-C16 alkyl or dialkylamino groups or further converted into amides.
  • the hydroxyl groups on the peptide side chain can be converted to C1-C16 alkoxy or C1-C16 esters using recognized techniques.
  • the phenyl and phenolic rings of the peptide side chain can be substituted with one or more halogen atoms such as F, Cl, Br, or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acid and its esters, or amides of these carboxylic acids.
  • the methylene group on the peptide side chain can be extended to a homologous C2-C4 alkylene group. Thiols can be protected with any of a variety of recognized protecting groups such as acetamide groups.
  • an embodiment of this application provides a nucleic acid molecule that encodes a mutant of the RSV pre-fusion F protein described in the first aspect.
  • Nucleic acid molecules, nucleic acids polymers composed of nucleotide units (ribonucleotides, deoxyribonucleotides, associated naturally occurring structural variants, and their synthetic non-natural analogs) linked by phosphodiester bonds, associated naturally occurring structural variants, and their synthetic non-natural analogs. Therefore, the term includes nucleotide polymers in which the nucleotides and the linkages therebetween include non-natural synthetic analogs such as, but not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA), etc.
  • PNA peptide-nucleic acid
  • oligonucleotide generally refers to short polynucleotides typically no more than about 50 nucleotides. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences in which "U” replaces "T” (i.e., A, U, G, C).
  • Nucleotide includes (but is not limited to) monomers comprising a base linked to a sugar (e.g., pyrimidine, purine, or synthetic analogues thereof) or monomers comprising a base linked to an amino acid (e.g., in peptide nucleic acids (PNA)).
  • a nucleotide is a monomer in a polynucleotide.
  • a nucleotide sequence refers to the base sequence in a polynucleotide.
  • Encoding refers to the inherent characteristics of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which acts as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the resulting biological characteristics.
  • a polynucleotide such as a gene, cDNA, or mRNA
  • nucleic acids encode mutants of this application.
  • Polynucleotides encoding polypeptides that include sequences degenerate due to the genetic code For example, polynucleotides encoding a disclosed antigen or an antibody that specifically binds to a disclosed antigen that includes a sequence degenerate due to the genetic code. There are 20 naturally occurring amino acids, most of which are designated by more than one codon. Therefore, all degenerate nucleotide sequences are included, provided that the amino acid sequence of the antigen or antibody that binds to the antigen encoded by the nucleotide sequence remains unchanged. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide.
  • the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Therefore, at each position within a protein-coding sequence that specifies arginine, the codon can be changed to any of the corresponding codons without altering the encoded protein.
  • Such nucleic acid variations are “silent variants,” a type of conserved variant.
  • Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variant.
  • each codon in a nucleic acid except AUG, which is typically the only codon for methionine
  • each "silent variant" of the nucleic acid encoding the polypeptide is implicit in each of the sequences.
  • it is codon-optimized for expression in mammalian cells and is operatively linked to a promoter.
  • Expression control sequence A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operatively linked.
  • An expression control sequence is operatively linked to a nucleic acid sequence when it controls and regulates transcription and, when appropriate, translation of the nucleic acid sequence. Therefore, an expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, splicing signals for introns, maintaining the appropriate reading frame of the gene to allow proper translation of the mRNA, and a stop codon.
  • control sequence is intended to include, at a minimum, components whose presence can affect expression, and may also include other components whose presence is advantageous, such as leader sequences and fusion chaperone sequences.
  • An expression control sequence may include a promoter.
  • a promoter is the smallest sequence sufficient to guide transcription. It also includes sequences sufficient to enable promoter-independent gene expression with respect to cell type specificity and histological characteristics. Those promoter elements that are tissue-specific and controllable or can be induced by external signals or agents; these elements may be located in the 5′ or 3′ region of a gene. This includes constitutive and inducible promoters (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as phage ⁇ , plac, ptrp, ptac (ptrp-lac heterozygous promoter), etc., can be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoters
  • promoters derived from the genome of mammalian viruses e.g., retroviral long terminal repeat sequences; adenovirus late promoters; vaccinia virus 7.5K promoters
  • retroviral long terminal repeat sequences e.g., adenovirus late promoters; vaccinia virus 7.5K promoters
  • Polynucleotides can be inserted into expression vectors containing promoter sequences that promote efficient transcription of the inserted genetic sequence in the host.
  • These expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow phenotypic selection in transformed cells.
  • RSVF proteins from different RSV subpopulations the nucleic acid sequences encoding these proteins, and the methods for manipulating and inserting these nucleic acid sequences into vectors are disclosed herein and known in the art (see, for example, Tan et al., PLOSone, 7: e51439, 2011; Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).
  • an embodiment of this application provides a carrier comprising the nucleic acid molecule described in the second aspect.
  • an engineered cell that expresses a mutant of the RSV pre-fusion F protein as described in the first aspect, or includes the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect.
  • Proteins are translated into proteins. Proteins can be expressed and retained inside cells, becoming components of the cell surface membrane, or secreted into the extracellular matrix or culture medium.
  • Engineered cell, host cell a cell in which the vector can reproduce and express its DNA.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the host cell. It should be understood that all progeny may differ from the parent cell because mutations can occur during replication. However, these progeny are included when the term "host cell" is used.
  • an embodiment of this application provides a method for producing a mutant of the RSV fusion-pre-fusion F protein, comprising the following steps:
  • the engineered cells described in the fourth aspect are cultured, and the mutant of the RSV fusion-pre-fusion F protein is isolated from the resulting culture supernatant.
  • an immune composition comprising a mutant of the RSV pre-fusion F protein as described in the first aspect or a nucleic acid molecule as described in the second aspect, and an immune adjuvant.
  • Immune adjuvants Mediators used to enhance antigenicity.
  • Adjuvants include suspensions of minerals (alum, aluminum hydroxide, or phosphate) adsorbed with antigens; or water-in-oil emulsions, for example, in which the antigen solution is emulsified in mineral oil (French incomplete adjuvant), sometimes including bactericidal mycobacteria (French complete adjuvant) to further enhance antigenicity (inhibiting antigen degradation and/or causing macrophage influx).
  • Immunostimulatory oligonucleotides e.g., those including CpG motifs
  • Adjuvants include biomolecules (“biological adjuvants”), such as co-stimulatory molecules.
  • exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF- ⁇ , IFN- ⁇ , G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and Toll-like receptor (TLR) agonists, such as TLR-9 agonists.
  • TLR Toll-like receptor
  • Adjuvants are well known to those skilled in the art (see, for example, Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed PreF antigen.
  • the immune adjuvants in the embodiments of this application are one or more of aluminum salt adjuvants, surfactants, polynucleotides, lipopolysaccharides, liposomes, and oil emulsion adjuvants.
  • aluminum salt adjuvants examples include Alum, CpG, Alum+CpG, MF59, AS04, AS01E, etc.
  • MF59 is a water-mixed adjuvant mainly composed of three parts: an oil phase, an emulsifier, and an excipient;
  • the oil phase is a mixture of micronized short-chain triglycerides suitable for human injection;
  • the emulsifier is a surfactant that allows the oil phase and water to mix uniformly;
  • the excipient mainly includes humectants and buffers such as glycerol, threitol, and ATP.
  • AS04 adjuvant is a mixture of AS03 adjuvant and MPL adjuvant.
  • AS03 adjuvant is a mixture of three surfactants: liposomes, TWEEN80, and SORBITAN.
  • AS01E is a nanoscale liposome solution prepared from DOPC, Chol, MPL, and QS-21.
  • the main component of the MPL adjuvant is lipopolysaccharide.
  • Immunogenic compositions Compositions comprising antigens that induce an immune response, such as a measurable CTL response against a virus expressing an antigen or a measurable B cell response (e.g., antibody production) against an antigen.
  • an immunogenic composition comprises one or more antigens (e.g., peptide antigens) or epitopes.
  • An immunogenic composition may also include one or more additional components capable of inducing or enhancing an immune response, such as excipients, carriers, and/or adjuvants.
  • an immunogenic composition is administered to induce an immune response that protects a subject from symptoms or illnesses induced by a pathogen.
  • an “immunogenic composition” includes... This includes recombinant RSVF proteins stabilized in their pre-fusion conformation, which induce measurable CTL responses against viruses expressing RSVF proteins, or induce measurable B cell responses against RSVF proteins (e.g., antibody production). It further refers to isolated nucleic acids encoding antigens, such as nucleic acids that can be used to express antigens (and thus to induce immune responses against such peptides).
  • the immunogenic composition may include an antigen or a nucleic acid encoding an antigen.
  • the immunogenic composition will typically include a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and/or other pharmaceutical agent.
  • a pharmaceutically acceptable carrier and/or other pharmaceutical agent for in vitro use, will typically include a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and/or other pharmaceutical agent.
  • the ability of any particular peptide, such as a disclosed RSVF protein stabilized in its pre-fusion conformation or a nucleic acid encoding a disclosed RSVF protein stabilized in its pre-fusion conformation, to induce a CTL or B cell response can be readily tested using recognized assays.
  • the immunogenic composition may include adjuvants well known to those skilled in the art.
  • Immunological reaction conditions include conditions that allow antibodies generated against a specific epitope to bind to said epitope and to a degree that is detectably greater than that of substantially all other epitopes and/or substantially exclude binding to substantially all other epitopes.
  • Immunological reaction conditions depend on the form of antibody binding reaction and are generally those used in immunoassay protocols or encountered in vivo.
  • the immunological reaction conditions used in the methods are “physiological conditions,” which include references to typical conditions (e.g., temperature, molar osmolality, pH) within living mammals or mammalian cells.
  • pH 7 e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5
  • water as the primary solvent
  • Molar osmolality is within the range supporting cell viability and proliferation.
  • an application is provided of a mutant of the RSV pre-fusion F protein described in the first aspect in the preparation of a respiratory syncytial virus antibody detection kit.
  • the respiratory syncytial virus antibody detection kit can be used to detect the corresponding antibodies.
  • the definition of the antibody is the same as in the first aspect, and it can be a neutralizing antibody or a binding antibody.
  • a respiratory syncytial virus antibody detection kit which includes a mutant of the RSV pre-fusion F protein described in the first aspect.
  • a ninth aspect of this application provides a method for preventing and treating lower respiratory tract infections caused by respiratory syncytial virus, comprising the following steps: administering a therapeutically effective amount of the immune composition described in the sixth aspect to a subject.
  • the composition is introduced into the subject via a chosen route.
  • Administration can be local or systemic.
  • the chosen route is intravenous
  • the composition is administered by introducing it into the subject's vein.
  • Effective amount An amount of an agent, such as the PreF antigen or a nucleic acid or other agent encoding the PreF antigen, sufficient to produce a desired response, such as an immune response against RSVF proteins, or to reduce or eliminate signs or symptoms of a symptom or disease, such as RSV infection. For example, this might be the amount required to inhibit viral replication or to measurably alter the external symptoms of a viral infection. Generally, this amount will be sufficient to measurably inhibit viral (e.g., RSV) replication or infectivity.
  • a dose that will reach a target tissue concentration (e.g., in respiratory tissue) that has been shown to achieve in vitro inhibition of viral replication is typically used.
  • an "effective amount” is the amount used to treat (including prevent) one or more symptoms and/or underlying causes of any condition or disease, such as the amount used to treat RSV infection. In one instance, an effective amount is a therapeutically effective amount. In one instance, an effective amount is the amount used to prevent the development of one or more signs or symptoms of a particular disease or symptom (e.g., one or more signs or symptoms associated with RSV infection).
  • prevention refers to the suppression of the full development of a disease or symptom in subjects at risk of developing a disease (e.g., RSV infection).
  • Treatment refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop.
  • improved in relation to a disease or pathological condition refers to any observable beneficial therapeutic effect.
  • a beneficial effect can be demonstrated, for example, by the delayed onset of clinical symptoms of the disease in susceptible subjects, a reduction in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or condition of the subject, or other parameters known in the art to be specific to a particular disease.
  • prophylactic treatment is a treatment administered to subjects who do not exhibit signs of disease or only exhibit early signs in order to reduce the risk of developing the lesion.
  • reduction is relative, meaning that a drug reduces a reaction or symptom if the reaction or symptom is quantitatively reduced after administration, or if it is reduced compared to a reference drug after administration.
  • prevention does not necessarily mean that the drug completely eliminates a reaction or symptom, provided that at least one characteristic of the reaction or symptom is eliminated.
  • immunogenic compositions that reduce or prevent infection or reaction can, but do not necessarily, completely eliminate such infection or reaction, provided that the infection or reaction is measurably reduced compared to an infection or reaction in the absence of the agent or compared to a reference agent, for example, by at least about 50%, such as at least about 70%, or about 80%, or even about 90% (i.e., reduced to 10% or less).
  • an embodiment of this application provides a method for detecting or separating RSVF-binding antibodies in a sample.
  • the method includes the following steps:
  • the mutant of the RSV pre-fusion F protein described in the first aspect is contacted with an RSVF-binding antibody in the sample to form an immune complex;
  • the immune complex is detected to detect or isolate RSV F-binding antibodies in a sample.
  • the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.
  • FIG. 1 illustrates the structure of the precursor polypeptide of wild-type RSV F protein, corresponding to the amino acid sequences SEQ ID NO.1 (human wild-type RSV A, STRAIN A2, 574aa), SEQ ID NO.2 (human wild-type RSV B, STRAIN 18537, 574aa), and SEQ ID NO.3 (bovine wild-type RSV B, STRAIN A51908, 572aa).
  • the precursor peptides include the signal peptide (aa1-25, as shown in SEQ ID NO. 7), the F2 peptide (aa26-109, as shown in SEQ ID NO. 5), the pep27 peptide (aa110-136, as shown in SEQ ID NO. 6), and the Fl peptide (aa137-574, as shown in SEQ ID NO. 4), with furin cleavage sites of RARR and KKRKRR.
  • the structural division of the naturally occurring full-length human RSV B is consistent with that of the naturally occurring full-length human RSV A2.
  • the natural full-length precursor peptide of bovine RSV B is 572aa in length, while the Fl peptide corresponds to aa137-572aa and contains furin cleavage sites RAKR and KKRKRR.
  • the ⁇ 3 helixes of the natural full-length human RSV A2 precursor polypeptide and the human RSV B precursor polypeptide are shown in SEQ ID NO. 8 and SEQ ID NO. 53, respectively.
  • the ⁇ 3 helix of the natural full-length bovine RSV B precursor polypeptide is also shown in SEQ ID NO. 53.
  • the ⁇ 3 sheets of the three natural full-length precursor polypeptides are all shown in SEQ ID NO. 9.
  • the native RSV F protein is initially expressed as the F0 polypeptide (precursor). After the F0 polypeptide is cleaved at its endoplasmic reticulum translocation signal peptide, it is processed by intracellular furin-like proteases at two sites. The pep27 polypeptide is cleaved, resulting in the mature F protein, which comprises an N-terminal F2 polypeptide and a C-terminal F1 polypeptide linked to the F2 polypeptide via two disulfide bonds. The mature F protein forms a trimer and is anchored to the cell membrane via a transmembrane domain mediated by the F1 polypeptide.
  • the RSV F protein undergoes an allosteric transformation from a pre-F conformation to a post-F conformation, during which the N-terminus and C-terminus of the F1 polypeptide are transformed into upward-pointing long ⁇ -helices.
  • Structural analysis revealed that introducing disulfide bonds between the ⁇ 3 helix and ⁇ 3 sheet yields a stable pre-F conformation.
  • FIG. 2 illustrates a schematic diagram of the structure of the stabilized mutant RSV F protein polypeptide monomer used for construction (amino acid sequence SEQ ID NO. 10).
  • This diagram corresponds to the natural mutant of the RSV/A2 subtype (amino acid sequence SEQ ID NO. 1), where the three natural mutations are located at P102A, I379V, and M447V, respectively.
  • other mutations involved in this application are designed based on this.
  • the mutants involved in the embodiments of this application are all based on this amino acid mutation.
  • the pep27 polypeptide is replaced by GS, is not affected by furin protease, and the transmembrane domain is deleted.
  • a His-Tag is added to the C-terminus of the protein to facilitate purification. After expression, this type of mutant exists in the cell culture supernatant as a soluble monomer.
  • FIG 3 illustrates the structural schematic of the stable mutant RSV F protein polypeptide trimer used for construction (amino acid sequence SEQ ID NO. 11).
  • the pep27 peptide is replaced by GS and is unaffected by furin protease.
  • the mutant RSV F protein of this application forms a trimer to mimic the native trimer state of the F protein; therefore, the trimerizing motif (T4foldon) of phage T4 foldon is fused to its C-terminus, and a purification tag is added to this C-terminus. After expression, this mutant exists as a soluble trimer in the cell culture supernatant.
  • Figure 4 illustrates that the native RSV F0 precursor protein or mature F protein contains seven pairs of disulfide bonds, which play a crucial role in stabilizing the structural features of the RSV F protein.
  • the F2 and F1 peptides are linked together by two pairs of disulfide bonds (C37/C439; C69/C212), which are essential for stabilizing the RSV pre-F apex.
  • Epitopes play a crucial role.
  • the remaining five disulfide bonds are distributed throughout the F1 polypeptide to help stabilize the unique structural features of the F protein.
  • This application involves mutations at other sites while maintaining the native disulfide bonds of the RSV F protein, thereby preserving its pre-fusion conformation (pre-F) without altering its basic structure.
  • Figure 5 illustrates a schematic diagram of the three-dimensional structure of the RSV F protein monomer (left) and a magnified schematic diagram of the ⁇ 3 and ⁇ 3 sheets (right).
  • the schematic diagram shows that the ⁇ 3 and ⁇ 3 sheets are antiparallel and closely spaced.
  • the RSV pre-F conformation is metastable and undergoes irreversible rearrangement to mediate membrane fusion or a nonfunctional post-F conformation triggered spontaneously.
  • a typical feature of post-F formation is the formation of a long coiled helix bundle through allosteric changes in the ⁇ 2, ⁇ 3, ⁇ 3, ⁇ 4, ⁇ 4, and ⁇ 5 sequences.
  • Figure 6 illustrates the amino acids involved at the ⁇ 3 and ⁇ 3 positions in this application, and the interatomic distance between the two amino acids that readily form disulfide bonds.
  • the amino acid sequence of the mutant was determined according to the mutation strategy, and the corresponding nucleic acid sequence was codon-optimized.
  • the optimized codons were conducive to expression in Chinese hamster ovary cells (Cricetulus griseus, CHO cells). Finally, it was ligated into the pcDNA3.1 vector via BamHI and XhoI restriction enzyme sites.
  • the ClonExpress II Recombinant Cloning Kit (Novizan Biotechnology Co., Ltd.) was used to perform vector substitution, insertion, and deletion operations.
  • Polymerase chain reaction (PCR) was used to amplify the fragments at both ends of the mutation site using the high-fidelity enzyme Phanta Max (Novizan Biotechnology Co., Ltd.). After gel recovery, the two fragments were fused by PCR. The recovered fusion fragment was homologously recombined into the pcDNA3.1 vector, which had been double-digested with BamHI and XhoI.
  • the constructed point mutation vector was sequenced and confirmed to be error-free by Beijing Qingke Biotechnology Co., Ltd.
  • the cloning vector was inoculated into 300 mL of LB (Amp+) medium and incubated at 37°C and 180 rpm for 16 h. Plasmids were then extracted using a large-scale/large-scale plasmid extraction kit and finally stored in 1 mL of sterile TE buffer. All commercial kits and reagents were performed according to the manufacturer's instructions.
  • the mutant protein was expressed using the ExpiCHOTM expression system (Thermofisher). Transient expression was performed strictly according to the manufacturer's standard protocol. In short, one day before transfection (Day – 1), ExpiCHO-STM culture was seeded to a final density of 3 ⁇ 106 –4 ⁇ 106 viable cells/mL. The following day (Day 0), viable cell density and survival percentage were measured. The cell density should reach approximately 7 ⁇ 106 –10 ⁇ 106 viable cells/mL. Survival rate should be 95–99% before proceeding with transfection. Cells were diluted to a final density of 6 ⁇ 106 viable cells/mL using fresh, preheated (37°C) ExpiCHOTM expression medium.
  • the culture flask was gently shaken to mix the cells, and any remaining cells were discarded.
  • the ExpiFectamineTM CHO/plasmid DNA complex was prepared using cold reagents (4°C) following the instructions in the reagent package insert. After inverting and mixing, add the ExpiFectamineTM CHO/DNA complex to the shake flask and incubate the cells on a shaker (8% CO2 , 37°C, 120 rpm). The day after transfection (Day 1, 18–22 hours post-transfection), add ExpiFectamineTM CHO enhancer and ExpiCHOTM adjuvant. Terminate the culture on day 8 post-transfection according to the standard experimental protocol and collect samples for analysis.
  • Protein purification was performed in two steps using affinity chromatography and ion exchange chromatography. Generally, the expression product was centrifuged (8000 rpm, 20 min) to harvest the culture supernatant and remove cells and cell debris. The culture supernatant was then filtered through a 0.45 ⁇ m filter to remove impurities. The processed supernatant was added to a Ni-Sepharose 6FF column (Cytiva) equilibrated with 25 mM Tris-HCl and 0.15 M NaCl (Buffer A; pH 8.0).
  • the column was then further washed with 25 mM Tris-HCl and 150 mM NaCl (pH 8.0) until A280 returned to baseline, followed by elution with 0–100% elution buffer (Buffer A). Elute linearly with elution buffer (25mM Tris-HCl, 150mM NaCl, 500mM imidazole, pH 8.0), collect the eluent, and use equilibration buffer as a replacement medium to remove imidazole.
  • the purified recombinant protein was detected by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis).
  • SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
  • the purified sample was added to a sample buffer containing SDS and a reducing agent (such as dimercaptoethanol or mercaptoacetic acid), treated in a boiling water bath, and then loaded onto the gel. After electrophoresis, the gel was stained with Coomassie blue, and images of the gel were acquired using a clear scanner or protein imaging system.
  • a reducing agent such as dimercaptoethanol or mercaptoacetic acid
  • Figure 7 illustrates the SDS-PAGE identification results of mutant monomers and trimers based on the ⁇ 3/ ⁇ 3 position mutation. Since the mutants differ from the wild-type F protein only in amino acid substitution, the molecular weights of the different mutant proteins are essentially the same, resulting in identical SDS-PAGE appearances. Therefore, JW-05 represents the mutant protein monomer. Similarly, JW-05-T4 represents the mutant trimer. The results show that both the monomers and trimers are slightly larger than the theoretical molecular weights (51 kDa/54 kDa), possibly due to glycosylation in the mutants.
  • the purity of the mutant protein was analyzed by HPLC using a Thermo U3000 high-performance liquid chromatograph and a Thermo TSKgel UP-SW2000 size exclusion column (TOSOH).
  • the mobile phase was 20 mM PBS buffer, the flow rate was 1 mL/min, and the elution composition was detected at 280 nm.
  • HPLC was used to determine the purity and homogeneity of the target protein.
  • Figure 8 illustrates the HPLC detection results of the mutant monomer and trimer.
  • Figure 8a shows the characteristic peak of the JW-05 monomer protein at 18.731 min.
  • Figure 8b shows the characteristic peak of the trimer JW-05-T4 at 15.958 min, with the target protein being singular and no protein aggregation observed.
  • RSV pre-F mutants were quantified using a double-antibody sandwich ELISA method.
  • Palivizumab which can simultaneously recognize pre-F, post-F, and F proteins, was used as the coating antibody to specifically recognize RSV pre-F.
  • the horseradish peroxidase-labeled specific monoclonal antibody D25 was used as the detection antibody (D25-HRP).
  • Standard curves were established using purified pre-F monomeric and trimer proteins as calibrators. The content of the sample to be tested was obtained by measuring its absorbance at OD450 and the dilution factor, and then converted from the standard curve. This method can detect the expression level of mutant pre-F protein under transient transfection and the residual content of pre-F mutant protein in the mutant thermostability test.
  • the sample detection procedure is as follows: The monoclonal antibody was diluted to 1 ⁇ g/L with carbonate buffer and coated onto an ELISA plate (Corning 9018). 100 ⁇ L per well, incubate at 37°C for 1 hour, then incubate overnight at 2–8°C; discard the liquid in the 96-well plate, wash 3 times with 20 mM PBS, then add 200 ⁇ L of blocking buffer (2% bovine serum albumin, component V) to each well, and block at room temperature for 60 min; aspirate the blocking buffer from the wells, wash 3 times with 20 mM PBS-T solution, add a series of 3-fold dilutions of the mutated pre-fusion conformation of F protein to the first row of wells of the 96-well microplate, with PBS as the negative control, incubate at 37°C for 60 min, aspirate the blocking buffer from the wells, and wash 3 times with 20 mM PBS-T solution; take anti-HIS-HRP conjugate, di
  • Figure 9 shows the expression level of Pre-F protein in the cell culture supernatant after transient transfection of monomeric RSV pre-F mutant protein into Expi-CHO cells.
  • the results showed that relatively low levels of pre-F protein could also be detected in the culture supernatant of transiently transfected F protein (WT).
  • WT transiently transfected F protein
  • the mutant When a cysteine residue was introduced at the ⁇ 3/ ⁇ 3 position of the F0 protein, the mutant significantly increased the pre-F protein content due to the formation of a disulfide bond bridge, indicating that the disulfide bond formed at the ⁇ 3/ ⁇ 3 position prevented the conformational change of the F protein to post-F, thus maintaining the F protein in its pre-fusion conformation.
  • Figure 10 shows the percentage of Pre-F protein remaining after heat treatment at 50°C for 1 h, 2 h, and 3 h in a pH 7.4 buffer system containing 20 mM PB and 150 mM NaCl at an antigen concentration of 0.1 mg/mL.
  • mice were immunized with different mutant proteins.
  • Female Balb/c mice 14–16 g were immunized with 5 ⁇ g of vaccine antigen mixed with aluminum hydroxide as an adjuvant.
  • An RSV inactivated vaccine (FI-RSV) group served as a control.
  • Intramuscular injections were administered at weeks 0 and 4 (28 days). Serum was collected 2 weeks after the second injection (42 days) to determine total IgG antibody titers and neutralizing antibody titers.
  • Immunization groups are shown in the table below:
  • Total IgG antibody was detected using an indirect ELISA method.
  • Pre-coated Pre-F protein was placed in 96-well plates. After blocking, the test serum was added and serially diluted, starting at an 800-fold dilution and followed by tri-fold dilutions. After washing to remove unbound serum, HRP-labeled goat anti-mouse secondary antibody was added and incubated. After incubation, unbound secondary antibody was washed away, and substrate was added for color development.
  • the mouse serum titer was determined by detecting absorbance at 450 nm and 630 nm. The dilution titer was calculated based on the serum dilution factor of the last well to which the absorbance was greater than the cut-off value.
  • mice were immunized with different mutant trimer proteins supplemented with aluminum hydroxide adjuvant, with an inactivated vaccine serving as a control.
  • the pre-F mutant trimer was used as the coating antigen to detect the level of antigen-specific IgG antibodies in mouse serum.
  • Neutralizing antibody titers were detected using the plaque assay.
  • a monolayer of Hep2 cells was prepared in 24-well plates. Serum was heat-inactivated in a 56°C water bath for 30 min. After inactivation, the serum was cooled to room temperature and diluted with serum-free EMEM medium at the following ratios (1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048). 100 ⁇ L of 50 pfu virus solution was mixed with an equal volume of serum diluent and incubated at 37°C for 1 h. The culture medium in the ppC VBNM plate was removed, and the plate was washed twice with sterile PBS.
  • FIG 17 shows the results of neutralizing antibody detection in the mutant protein and the RSV inactivated vaccine (FI-RSV). This indicates that the mutant trimer has good immunogenicity and can induce high levels of neutralizing antibodies against RSV A2 type in mice, with significantly higher levels than the inactivated vaccine group.
  • Figure 18 shows the adsorption rate of neutralizing antibodies in serum. The results show that serum without antigen adsorption can completely neutralize the virus, and no cytopathic effect occurs. In serum after adsorption of antigens from various mutants, neutralizing antibodies were completely cleared, and the serum could not prevent viral infection of cells, resulting in typical cytopathic effects. This result indicates that each mutant has the same neutralizing epitope and can be recognized by antibodies in naturally infected serum, which is the basis for its development as a candidate vaccine based on RSV pre-F.
  • JW-01 to JW-06 in Example 1 corresponding mutants JW-01-18537 to JW-06-18537 and JW-01-51908 to JW-06-51908 were designed, starting with the F protein precursor polypeptide of human RSV STRAIN 18537 and bovine RSV STRAIN A51908, respectively, as shown in the table below.
  • the mutant content (denoted as V) was detected using the double-antibody sandwich method as described in Example 1. Further, the samples with the previously determined content were divided into two groups for treatment (one group was placed at 4°C for 4 weeks, and the other group at 50°C for 3 hours). The mutant content in the samples was then detected again (denoted as V’), and V’/V ⁇ 100% was calculated. The results are shown in the table below.
  • Table 7 describes the effects of introducing disulfide bonds into the ⁇ 3 helix and ⁇ 3 sheet of the F protein of different RSV subtypes on the expression level and stability of mutants. The results show that because the F protein of different RSV subtypes is relatively conserved, the introduction of disulfide bonds into the ⁇ 3 helix and ⁇ 3 sheet has similar effects on the expression level and stability of the F protein of different RSV subtypes, and all can express the preF protein to varying degrees.
  • the mutant content (denoted as V) was detected using the double-antibody sandwich method as described in Example 1. Further, the samples with the previously determined content were divided into two groups for treatment (one group was placed at 4°C for 4 weeks, and the other group at 50°C for 3 hours). The mutant content in the samples was then detected again (denoted as V’), and V’/V ⁇ 100% was calculated. The results are shown in Table 9 below.
  • Table 9 describes the expression levels and thermostability of mutant proteins obtained by linking different flexible short peptides to the p27 position of the ⁇ 3 helix and ⁇ 3 sheet mutant F proteins. The results show that the thermostability of mutant proteins obtained with different flexible short peptide lengths and linking positions varies slightly, but all can be expressed normally.

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Abstract

La présente invention concerne un mutant d'une protéine F de pré-fusion du VRS, sa préparation et son utilisation. L'invention concerne un mutant de la protéine F de pré-fusion du VRS. Un polypeptide F1 de celui-ci a un substituant cystéine 1 dans une région d'hélice α3, et un substituant cystéine 2 dans une région de feuille β3. Le substituant cystéine 1 et le substituant cystéine 2 forment une liaison disulfure.
PCT/CN2024/096123 2024-05-22 2024-05-29 Mutant de protéine f de pré-fusion de vrs, sa préparation et son utilisation Pending WO2025241209A1 (fr)

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