WO2023208206A1 - Mutant de protéine f du vrs et son utilisation - Google Patents

Mutant de protéine f du vrs et son utilisation Download PDF

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WO2023208206A1
WO2023208206A1 PCT/CN2023/091647 CN2023091647W WO2023208206A1 WO 2023208206 A1 WO2023208206 A1 WO 2023208206A1 CN 2023091647 W CN2023091647 W CN 2023091647W WO 2023208206 A1 WO2023208206 A1 WO 2023208206A1
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protein
combination
mutant
mutation
amino acid
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PCT/CN2023/091647
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English (en)
Chinese (zh)
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王弈
毕帅
许铮
刁云珍
单栢强
李丹丹
万季
赵钊
潘有东
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北京新合睿恩生物医疗科技有限公司
深圳市新合生物医疗科技有限公司
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Priority to CN202380011931.9A priority Critical patent/CN117715923A/zh
Publication of WO2023208206A1 publication Critical patent/WO2023208206A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
    • C07K14/135—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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedical technology, and in particular to an RSV F protein mutant and its application.
  • Respiratory syncytial virus (RSV) infection causes substantial morbidity and mortality in infants, young children, and the elderly. There are an estimated 64 million infections and 160,000 deaths worldwide each year. Pneumonia caused by RSV infection is the leading cause of clinical death. The primary cause of cases. To date, passive prophylaxis with monoclonal antibodies has been limited in part to high-risk infants due to its modest efficacy. There are currently no safe and effective vaccines approved for RSV infection, highlighting the urgent need for RSV vaccines that induce or provide protective immune responses.
  • RSV expresses two major surface glycoproteins: fusion protein (F protein) and adhesion protein (G protein). Both play an integral role during RSV infection and both have been shown to induce protective neutralizing antibody responses and are major protective antigens. Since the amino acid sequence of F protein is approximately 90% conserved among RSV subtypes related to human infection, its genetic stability is much higher than that of G protein. In addition, F protein is an important target for inducing the body to produce CD8+ T cells. Therefore, RSV F protein is an important target for the development of RSV vaccines.
  • F protein fusion protein
  • G protein adhesion protein
  • RSV enters cells through F protein-mediated fusion of viral particles with infected cell membranes, and the F protein abundantly expressed on the surface of infected cells mediates the fusion of adjacent cells to form a syncytium.
  • the full-length sequence of wild-type F protein has 574 amino acids, and the amino acid sequence is shown in SEQ ID NO:1. Among them, amino acids 1-513 are the extracellular region of F protein, and amino acids 514-574 are the transmembrane region and intracellular region of F protein.
  • the mature RSV F protein initially exists in a metastable pre-fusion (pre-fusion F) conformation and subsequently undergoes conformational changes, resulting in the insertion of the hydrophobic fusion peptide into the host cell membrane.
  • post-fusion F elongated post-fusion conformation
  • the prefusion conformation F protein Due to the inherent instability of the prefusion conformation of the F protein, the prefusion conformation F protein has the tendency to trigger prematurely into a conformationally stable postfusion conformation F form both in solution and on the virus surface.
  • Current research has achieved stabilization of the pre-fusion conformation F through protein engineering, and the stabilized fusion in animal models Prefusion conformation F induces higher titers of neutralizing antibodies than postfusion conformation F.
  • the stabilization of the pre-fusion conformation F is mainly achieved through the formation of disulfide bonds between different amino acids in the molecule and the mutation of amino acids in the hydrophobic cavity position.
  • the current stabilizing mutation scheme for the pre-fusion conformation F suffers from the problems of low protein expression and insufficient stability of the pre-fusion conformation.
  • the present application provides a mutant of the wild-type RSV F protein, which significantly increases the protein expression of the pre-fusion conformation F and has increased stability of the pre-fusion conformation.
  • the present application provides a mutant of wild-type RSV F protein, wherein the mutant includes at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, and the amino acid mutation is:
  • the cavity-filling mutation includes: the amino acid at position 190 is replaced by F;
  • the engineered disulfide bond mutations include substitution of amino acids at positions 466 and 443 with C;
  • Position numbering is based on the sequence shown in SEQ ID NO:1.
  • the cavity-filling mutation further includes:
  • amino acid at position 54 is replaced by H; and/or
  • amino acid at position 207 or 296 is replaced by L or I.
  • the electrostatic mutation includes:
  • the amino acid at position 486 was substituted with S.
  • the amino acid mutation further includes: the amino acid at position 215 is replaced by P.
  • the mutant is in the form of a trimer.
  • the mutant has a higher expression level of the pre-fusion F protein than the wild-type RSV F protein and has increased stability of the pre-fusion conformation.
  • the wild-type RSV is subtype A or subtype B.
  • amino acid mutation is a combination selected from the following mutations:
  • the mutant of the wild-type RSV F protein comprises a sequence such as any one of SEQ ID NO:7, 8, 14-28, and 29-30 or consists of a sequence such as SEQ ID NO:7, 8, Composed of any sequence from 14-28 and 29-30.
  • the mutant of the wild-type RSV F protein comprises a cell sequence consisting of amino acid sequences 1-513 of any one of SEQ ID NO: 7, 8, 14-28, and 29-30. outer area.
  • the mutant of the wild-type RSV F protein is composed of the amino acid sequence 1-513 of any one of SEQ ID NO: 7, 8, 14-28 and 29-30.
  • the extracellular region and the transmembrane and intracellular regions of the wild-type F protein are the full-length F protein.
  • the present application also provides an extracellular region polypeptide of a wild-type RSV F protein mutant.
  • the amino acid sequence of the extracellular region polypeptide includes any of SEQ ID NOs: 7, 8, 14-28 and 29-30.
  • Amino acids 1-513 of a sequence may be composed of amino acids 1-513 of any sequence such as SEQ ID NO: 7, 8, 14-28 and 29-30.
  • the present application provides a nucleic acid molecule encoding the mutant described above.
  • the present application provides an expression vector or cell, which contains the nucleic acid molecule described above.
  • the present application provides a kit for immunizing human subjects against viral infection, which includes the mutant described above.
  • the viral infection is RSV infection.
  • the present application provides a pharmaceutical composition, which contains the above-mentioned mutant or the above-mentioned nucleic acid molecule or the above-mentioned expression vector or cell and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine.
  • the present application provides the use of the pharmaceutical composition described above for inducing an immune response against RSV infection.
  • the present application provides a method for preventing or treating RSV infection, comprising administering to a subject an effective amount of the above-mentioned mutant or the above-mentioned pharmaceutical composition.
  • This application provides the use of the above-mentioned mutant or the above-mentioned pharmaceutical composition in the preparation of drugs or vaccines for preventing or treating RSV infection.
  • the mutant described in this application can significantly increase the protein expression of the pre-fusion conformation F, and has increased stability of the pre-fusion conformation.
  • Figures 1A to 1C are schematic diagrams of the expression levels of F proteins of different conformations expressed by recombinant plasmids of each mutant.
  • Figure 1A is a schematic diagram of the expression levels of F proteins of pre-fusion conformation expressed by recombinant plasmids of each mutant.
  • Figure 1B is A schematic diagram showing the expression level of the pre-fusion conformation and trimerized F protein expressed by the recombinant plasmid of each mutant.
  • Figure 1C is a schematic diagram showing the expression level of the post-fusion conformation F protein expressed by the recombinant plasmid of each mutant.
  • FIGS 2A to 2C are schematic diagrams showing the amount of pre-fusion conformation F protein recognized by pre-fusion conformation antibody D25 expressed by different mutants.
  • Figure 3 is a schematic diagram showing the expression levels of F protein in pre-fusion conformation expressed by different mutants.
  • Figure 4 is a schematic diagram showing the expression levels of F protein in pre-fusion conformation expressed by different mutants.
  • Figure 5 is a schematic diagram of ELISA detection of the stability of pre-fusion conformation F protein in cell culture supernatant.
  • Figures 6A to 6D are schematic diagrams of the expression patterns of each neutralizing epitope of the recombinant plasmid encoding the mutant expressing the pre-fusion conformation F protein.
  • Figure 6A is a schematic diagram of the neutralizing epitope expression of the pre-fusion conformation F protein expressed by the recombinant plasmid encoding the mutant.
  • Figure 6B is a schematic expression map of the recombinant plasmid encoding the mutant expressing the neutralizing epitope (Site IV and Site V) of the pre-fusion conformation trimer F protein
  • Figure 6C is a recombinant plasmid encoding the mutant
  • Figure 6D is the expression map of the neutralizing epitope (Site V) of the pre-fusion conformation F protein expressed by the recombinant plasmid encoding the mutant.
  • Figure 7 is a schematic diagram of the recombinant plasmid encoding the mutant.
  • Figures 8A to 8B are schematic diagrams of flow cytometry to detect the mean fluorescence intensity (MFI) of pre-fusion conformation F protein expressed on the cell surface combined with antibody D25 or antibody AM14.
  • MFI mean fluorescence intensity
  • Figure 8A is a schematic diagram of the average fluorescence intensity of the pre-fusion conformation F protein expressed on the cell surface combined with the antibody D25
  • Figure 8B is a schematic diagram of the average fluorescence intensity of the pre-fusion conformation trimer F protein expressed on the cell surface combined with the antibody AM14.
  • Figures 9A to 9B are schematic diagrams of immunizing BALB/c mice with wild-type and mutant F protein mRNA-LNP vaccines to induce serum F protein-specific binding antibody IgG and immunizing BALB/c mice to induce serum neutralizing antibodies
  • Figure 9A is a schematic diagram of wild-type and mutant F protein mRNA-LNP vaccines immunizing BALB/c mice to induce serum F protein-specific binding antibodies
  • Figure 9B is a schematic diagram of wild-type and mutant F protein mRNA-LNP vaccines immunizing BALB/c mice. Schematic representation of induction of serum neutralizing antibodies.
  • mutation refers to the deletion, addition or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide.
  • annotation “(amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein)” is used to replace an amino acid at the first specific position in the protein sequence.
  • S466C means that the serine (S) residue at position 466 of the amino acid sequence of the reference protein is replaced by a cysteine (C) residue (in the mutant of the reference protein).
  • wild-type protein, sequence or polypeptide refers to a naturally occurring protein, sequence or polypeptide that has not been artificially modified by selective mutation.
  • immunogenicity refers to the ability of a substance to elicit, prime, stimulate or induce an immune response in an animal against a specific antigen in the presence or absence of an adjuvant.
  • glycoprotein refers to a protein containing oligosaccharide chains (glycans) covalently attached to polypeptide side chains. Carbohydrates are attached to proteins in a co- or post-translational modification called glycosylation.
  • a glycan is a polysaccharide or oligosaccharide, which may also be used to refer to the carbohydrate portion of a glycoconjugate such as a glycoprotein, glycolipid or proteoglycan.
  • mutant of the wild-type RSV F protein refers to a polypeptide that has an introduced mutation relative to the wild-type F protein and is immunogenic against the wild-type F protein.
  • a pharmaceutically acceptable carrier refers to a material or composition that, when combined with the active ingredient, is compatible with the active ingredient and does not cause toxicity or other undesirable effects when administered to an individual, especially a mammal.
  • a pharmaceutically acceptable carrier can be a solvent, surfactant, suspending agent, buffer, lubricant, emulsifier, absorbent, dispersion medium, coating and/or stabilizer.
  • prefusion-specific antibody refers to an antibody that specifically binds into a prefusion conformation An antibody that binds to the RSV F glycoprotein but does not bind to the RSV F protein in a post-fusion conformation. In some embodiments, the prefusion-specific antibody is D25.
  • immune response refers to any detectable response of one or more cells of the host mammal's immune system to a stimulus, such as an immunogen, including but not limited to innate immune responses (e.g., Toll receptor signaling transduction cascade), cell-mediated immune responses (e.g., T cells of the immune system (such as antigen-specific T cells) and non-specific cell-mediated responses), and humoral immune responses (e.g., B cell-mediated reactions, such as production of antibodies and secretion into plasma, lymph and/or tissue fluid).
  • innate immune responses e.g., Toll receptor signaling transduction cascade
  • cell-mediated immune responses e.g., T cells of the immune system (such as antigen-specific T cells) and non-specific cell-mediated responses
  • humoral immune responses e.g., B cell-mediated reactions, such as production of antibodies and secretion into plasma, lymph and/or tissue fluid.
  • immune responses include changes (e.g., increases) in Toll-like receptor activation, lymphokine (e.g., cytokines (e.g., Th1, Th2, or Th17-type cytokines) or chemokines) expression or secretion, macrophages Activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cells) activation, NK cell activation, B cell activation (e.g., antibody production and/or secretion), immunogen (e.g., antigen (e.g., immunogen binding of peptides) to MHC molecules, induction of cytotoxic T lymphocyte ("CTL") responses, induction of B cell responses (e.g., antibody production), and expansion of cells of the immune system (e.g., T cells and B cells) (e.g., growth of a cell population), and increased antigen processing and presentation by antigen-presenting cells.
  • lymphokine e.g., cytokines (e.g
  • an antibody binds a given target molecule
  • the term "specifically binds" means that the antibody binds to the target molecule with a higher affinity than it binds to other substances tested, e.g., specifically binds in a prefusion conformation
  • An antibody to the RSV F protein is an antibody that binds to the RSV F protein in the pre-fusion conformation with a higher affinity than it binds to the RSV F protein in the post-fusion conformation.
  • vaccine refers to a pharmaceutical composition containing an immunogen capable of eliciting a prophylactic or therapeutic immune response in an individual.
  • a vaccine elicits an antigen-specific immune response against a pathogen, such as a viral pathogen.
  • expression vector refers to a vector capable of expressing an insert in a target cell and typically containing control sequences that drive expression of the insert, such as enhancer, promoter, and terminator sequences.
  • Foreign nucleic acid molecules are called "inserts.”
  • Expression vectors usually consist of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or source of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus (Ad) vectors), and artificial chromosomes.
  • conservative substitution refers to the replacement of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. The following 6 groups each contain amino acids that are conservative substitutions for each other:
  • the term "effective amount” refers to an amount of an agent sufficient to produce a desired response. For example, this may be an amount required to inhibit viral replication or measurably alter the outward symptoms of viral infection.
  • the term "host cell” refers to a cell in which a vector can be propagated and its DNA or RNA expressed, and the cell may be prokaryotic or eukaryotic.
  • Optimal alignment of sequences for comparison can be performed by, for example, the local homology algorithm, Smith and Waterman, Adv. Appl. Math. 2:482, 1981; the homology alignment algorithm, Needleman and Wunsch , Mol. Biol. 48:443, 1970; Similarity search methods, Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988; Computerized implementation of these algorithms (in the Wisconsin Genetics software package GAP, BESTFIT, FASTA, and TFASTA, Genetics Computer Group, 575 Science Dr., Madison, WI); or manual comparison and visual inspection (see, e.g., Sambrook et al.
  • the present application provides a mutant of the wild-type RSV F protein, wherein the mutant includes at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, and the amino acid mutation is at least one engineered dipeptide. a combination of a sulfide mutation and at least one cavity-filling mutation or a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation and at least one electrostatic mutation;
  • the cavity-filling mutation includes: the amino acid at position 190 is replaced by F;
  • the engineered disulfide mutations include substitution of amino acids at positions 466 and 443 with C;
  • Position numbering is based on the sequence shown in SEQ ID NO:1.
  • the mutant has a higher expression level of the F protein in the prefusion conformation relative to the corresponding wild-type F protein and has increased stability of the prefusion conformation.
  • the RSV F protein is translated from mRNA into a single 574 amino acid polypeptide precursor (called “FO” or "FO precursor”).
  • the RSV F0 polypeptide includes an N-terminal signal peptide (amino acid residues 1-25), F2 polypeptide (amino acid residues 26-109), pep27 polypeptide (amino acid residues 110-136), and include the F1 extracellular domain (amino acid residues 137-524), transmembrane domain (525-550) and cytoplasmic tail F1 polypeptide (amino acid residues 551-574).
  • the "FO polypeptide” (F0) refers to the precursor polypeptide of the RSV F protein.
  • F1 polypeptide refers to the polypeptide chain of the mature RSV F protein.
  • Native F1 includes approximate residues 137-574 of the RSV F0 precursor and consists (from N-terminus to C-terminus) of the extracellular region (approximately residues 137-524), and the transmembrane domain (approximately residues 525-550) and a cytoplasmic domain (approximately residues 551-574).
  • F2 polypeptide refers to the polypeptide chain of the mature RSV F protein. Native F2 includes approximate residues 26-109 of the RSV F0 precursor.
  • the term includes native F2 polypeptides and F2 polypeptides derived from the native sequence that include modifications (e.g., amino acid substitutions, insertions, or deletions) that are, for example, designed to stabilize the F mutant or enhance the immunogenicity of the F mutant.
  • modifications e.g., amino acid substitutions, insertions, or deletions
  • the F2 polypeptide is linked to the F1 polypeptide through two disulfide bonds to form an F2-F1 heterodimer.
  • the RSV F protein consists of F1/F2 heterodimers that trimerize to form a multimeric protein.
  • glycoprotein refers to a protein containing oligosaccharide chains (glycans) covalently attached to polypeptide side chains. Carbohydrates are attached to proteins in a co- or post-translational modification called glycosylation.
  • glycosylation site refers to an amino acid sequence on the surface of a polypeptide, such as a protein, that accommodates the attachment of glycans. N- The linked glycosylation site is the triploid sequence of NX(S/T), where N is asparagine, X is any residue except proline, and (S/T) is serine or threonine acid residue.
  • Glycans are polysaccharides or oligosaccharides. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate such as a glycoprotein, glycolipid or proteoglycan.
  • the amino acid sequences of numerous native RSV F proteins from different RSV subtypes, as well as the nucleic acid sequences encoding such proteins, are known in the art.
  • the native RSV F protein exhibits significant sequence conservation among RSV subtypes. For example, RSV subtypes A and B have >90% sequence identity, and within RSV subtypes, the F0 sequence identity is even higher; for example, in RSV A, B-RSV F0 precursor proteins have approximately 98% sequence identity. Nearly all identified RSV F0 precursor sequences consist of 574 amino acids in length, with lengths often varying slightly due to the length of the C-terminal cytoplasmic tail. Sequence identity between various native RSV F proteins is known in the art (see, e.g., WO2014/160463).
  • RSV F sequences Given the substantial conservation of RSV F sequences, one of ordinary skill in the art can readily compare amino acid positions between different native RSV F sequences to identify corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, the furin cleavage site falls within the same amino acid position in nearly all identified native RSV F0 precursor proteins. Therefore, the conservation of the native RSV F protein sequence among strains and subtypes allows the use of a reference RSV F sequence to compare amino acids at specific positions in the RSV F protein.
  • the amino acid positions of the RSV F protein are referenced to the sequence of the F0 precursor polypeptide described in SEQ ID NO: 1 (the amino acid sequence of the full-length native F precursor polypeptide of the RSV A2 strain ).
  • SEQ ID NO: 1 the amino acid sequence of the full-length native F precursor polypeptide of the RSV A2 strain.
  • RSV FO sequences may have different numbering systems if, for example, additional amino acid residues are added or removed compared to SEQ ID NO: 1.
  • amino acid residue when a specific amino acid residue is referred to by its number, the amino acid residue is not limited to the amino acid at the position numbered according to SEQ ID NO: 1, but also encompasses all RSV F sequences that align with SEQ ID NO: 1 The corresponding/equivalent/corresponding amino acid residue obtained, even if the residue is not at the same exact numbering position, e.g. if the RSV sequence is shorter or longer than SEQ ID NO:1, or has an insertion or deletion compared to SEQ ID NO:1 Case.
  • amino acid sequence of SEQ IN NO:1 is as follows:
  • the "engineered disulfide bond mutation” refers to the mutation of a pair of amino acid residues in the wild-type RSV F protein into a pair of cysteine residues.
  • the introduced cysteine residue pairs allow the formation of a disulfide bond between the introduced cysteine residues.
  • the engineered disulfide bond mutations include substitution of amino acids at positions 466 and 443 with C, such as S466C and S443C.
  • the "cavity-filling mutation” refers to the existence of some cavities inside some wild-type RSV F proteins. Mutation of the amino acids near these cavities causes the size of these cavities to change or disappear. In some applications, such cavity-filling mutations help stabilize the prefusion conformation of RSV F protein mutants.
  • Amino acids to be substituted for cavity-filling mutations typically include small aliphatic amino acids (eg, Gly, Ala, and Val) or small polar amino acids (eg, Ser and Thr). It may also include amino acids that are embedded in a prefusion conformation but exposed to solvent in a postfusion conformation. Replacement amino acids may be large aliphatic amino acids (Ile, Leu and Met) or large aromatic amino acids (His, Phe, Tyr and Trp).
  • the mutants include at least one cavity-filling mutation. In some embodiments, the mutants include at least two cavity-filling mutations. In some embodiments, the cavity-filling mutation includes substitution of the amino acid at position 190 with an F (eg, S190F or N190F). In some embodiments, the cavity-filling mutation may also be: the amino acid at position 54 is replaced by H, such as T54H; and/or the amino acid at position 207 or 296 is replaced by L or I, such as V207L and V296L.
  • electrostatic mutations we mean amino acid mutations introduced into the wild-type RSV F protein that reduce the ionic repulsion between residues that are close to each other in the folded structure or increase the ionic attraction between the residues.
  • electrostatic mutations can be introduced to improve the stability of the prefusion conformation.
  • Unfavorable electrostatic interactions in the prefusion or prefusion trimer conformation can be identified by methods known in the art, such as by visual inspection of the crystal structure of RSV F in the prefusion or prefusion trimer conformation, or by using computational Protein design software (such as BioLuminate [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015.] and Rosetta [Rosetta, University of Washington, Seattle, 2015.]).
  • computational Protein design software such as BioLuminate [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015.] and Rosetta [Rosetta, University of Washington, Seattle, 2015.]).
  • the amino acid mutation further includes: the amino acid at position 215 is replaced by P, such as S215P.
  • the amino acid mutation is a combination of at least one engineered disulfide mutation and at least one cavity filling mutation or at least one engineered disulfide mutation, at least one cavity filling mutation and at least one electrostatic A combination of mutations or a combination of at least one engineered disulfide bond mutation, at least one cavity filling mutation, and substitution of the amino acid at position 215 with P.
  • the mutants are in trimer form.
  • the amino acid mutation is a combination of mutations selected from:
  • the mutants comprise SEQ ID NOs: 7, 8, 14-28, and 29-30 Any one or more sequences in SEQ ID NO: 7, 8, 14-28 and 29-30.
  • the RSV F protein mutants provided herein can be prepared by conventional methods known in the art, such as by expression in a recombinant host system using an appropriate vector.
  • Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria and yeast cells.
  • suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen )).
  • suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, which are typically transformed by spliced adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells and HeLa cells.
  • Suitable avian cells include, for example, chicken embryonic stem cells (eg, EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (eg, ELL-O), and duck cells.
  • Suitable insect cell expression systems such as baculovirus vector systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.
  • Suitable vectors for expression of recombinant proteins in insect or mammalian cells are well known in the art.
  • Suitable vectors may contain a variety of components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcription control elements (e.g., promoter, enhancer, terminator) and/or one or more translation signals; and for targeting to the secretory pathway in a host cell of choice (e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species) Signal sequence or leader sequence.
  • an origin of replication e.g., a selectable marker gene
  • expression control elements such as transcription control elements (e.g., promoter, enhancer, terminator) and/or one or more translation signals
  • a host cell of choice e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species
  • baculovirus expression vector such as pFastBac (Invitrogen)
  • Baculovirus particles are amplified and used to infect insect cells to express recombinant proteins.
  • vectors are used that will drive expression of the construct in the desired mammalian host cell (eg, Chinese hamster ovary cells).
  • RSV F protein mutant polypeptides can be purified using any suitable method.
  • methods for purifying RSV F protein mutant polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687(2004).
  • Suitable methods for purifying the desired protein are well known in the art and include precipitation and various types of chromatography, such as hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, chelation chromatography and size exclusion chromatography.
  • a suitable purification scheme can be generated using two or more of these or other suitable methods.
  • RSV F protein mutant polypeptides may include "tags" to aid purification, such as epitope tags or histidine (HIS) tags.
  • tags to aid purification, such as epitope tags or histidine (HIS) tags.
  • Such tagged polypeptides can be conveniently purified, for example from conditioned media by chelation chromatography or affinity chromatography.
  • the mutant comprises at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, the amino acid mutation being at least one engineered disulfide bond mutation and at least one cavity filling mutation.
  • the cavity-filling mutation further includes: the amino acid at position 54 is replaced by H; and/or the amino acid at position 207 or 296 is replaced by L or I.
  • the mutant comprises at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, the amino acid mutation being at least one engineered disulfide bond mutation and at least one cavity filling mutation.
  • the cavity-filling mutation further includes: the amino acid at position 54 is replaced by H; and/or the amino acid at position 207 or 296 is replaced by L or I.
  • the electrostatic mutation includes: the amino acid at position 486 is replaced with S.
  • the amino acid mutation further includes: the amino acid at position 215 is replaced by P.
  • the mutant comprises at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, the amino acid mutation being at least one engineered disulfide bond mutation and at least one cavity filling mutation.
  • the cavity-filling mutation further includes: the amino acid at position 54 is replaced by H substitution; and/or the amino acid at position 207 or 296 is substituted by L or I.
  • the electrostatic mutation includes: the amino acid at position 486 is replaced with S.
  • the amino acid mutation further includes: the amino acid at position 215 is replaced by P.
  • the mutants are in trimer form.
  • the mutant has a higher expression level of the prefusion F protein than the wild-type RSV F protein and has increased stability of the prefusion conformation.
  • the wild-type RSV is subtype A or subtype B.
  • the amino acid mutation is a combination of mutations selected from:
  • the wild-type RSV F protein uses the A2 strain of subtype A, and its sequence is shown in SEQ ID NO: 1.
  • mutants described in the present application have a higher level of pre-fusion protein expression and increased stability of the pre-fusion protein conformation.
  • nucleic acid molecules encoding the RSV F protein mutants described above. These nucleic acid molecules include DNA, cDNA and RNA sequences. Nucleic acid molecules can be incorporated into vectors, such as expression vectors. In some embodiments, the nucleic acid molecule encodes a precursor FO polypeptide that, when expressed in an appropriate cell, is processed into an RSV F mutant of the present application. In some embodiments, the nucleic acid molecule encodes a precursor FO polypeptide that when expressed in an appropriate cell is processed into an RSV F mutant, wherein the precursor FO polypeptide includes a signal peptide from the N-terminus to the C-terminus, F2 polypeptide, Pep27 polypeptide and F1 polypeptide.
  • the nucleic acid molecule encodes a mutant selected from:
  • the amino acids at key mutation positions 466 and 443 were substituted with C.
  • the nucleic acid molecule encodes a mutant in which the cavity-filling mutation is such that the amino acid at position 54 is replaced by H; and/or the amino acid at position 207 or 296 is replaced by L or I.
  • the nucleic acid molecule encodes a mutant in which the electrostatically mutated amino acid at position 486 is replaced with S.
  • the nucleic acid molecule encoding amino acid mutations also includes a mutant in which the amino acid at position 215 is replaced by P.
  • the nucleic acid molecule encodes a mutant selected from:
  • the present application provides an expression vector comprising the nucleic acid molecule described above.
  • the present application provides a kit for immunizing human subjects against viral infection, which includes the mutant described above.
  • the present application provides a pharmaceutical composition, which contains the above-mentioned mutant or the above-mentioned nucleic acid molecule or the above-mentioned expression vector and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine.
  • the pharmaceutical composition further includes an immunomodulatory agent, such as an adjuvant.
  • suitable adjuvants include aluminum salts, such as aluminum hydroxide and/or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90/ 14837); saponin preparations such as, for example, QS21 and immunostimulatory complexes (ISCOMS) (see, for example, US Patent No.
  • bacteria or Microbial derivatives examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif-containing oligonucleotides, ADP-ribosylated bacterial toxins or mutations thereof bodies, such as Escherichia coli heat-labile enterotoxin LT, cholera toxin CT or lipid nanoparticles.
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • CpG-motif-containing oligonucleotides such as Escherichia coli heat-labile enterotoxin LT, cholera toxin CT or lipid nanoparticles.
  • the vector may also be used by using a heterologous nucleic acid encoding a fusion of the oligomerization domain of C4 binding protein (C4bp) with an antigen of interest.
  • body adjuvants eg Solabomi et al., 2008, Infect Immun 76:3817-23.
  • the compositions include aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or combinations thereof, at a concentration of 0.05 mg to 5 mg per dose (eg, 0.075 mg to 1.0 mg) aluminum content.
  • the application provides a method of inducing an immune response against RSV in a subject, comprising administering to the subject an effective amount of an RSV F protein mutant, a nucleic acid molecule encoding an RSV F protein mutant, or An expression vector containing a nucleic acid molecule.
  • the subject is a human.
  • the human is a child, such as an infant.
  • the human is a female, particularly a pregnant female.
  • the pharmaceutical composition can be administered to a subject with or without the administration of an adjuvant.
  • An effective amount administered to a subject is an amount sufficient to elicit an immune response in the subject against an RSV antigen, such as RSV F protein.
  • Subjects that may be selected for treatment include those who are at risk of developing RSV infection due to exposure or potential exposure to RSV. Since nearly all humans are infected with RSV by the age of 2 years, the entire birth cohort was included as an immune-related group.
  • Subjects at greatest risk for RSV infection with severe symptoms include children with prematurity, bronchopulmonary dysplasia, and congenital heart disease.
  • Administration of the pharmaceutical compositions provided herein can be carried out using standard routes of administration.
  • Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.
  • the total dose of the pharmaceutical composition provided to the subject during an administration may vary, as dosage variations are known to the skilled artisan.
  • One or more booster administrations of one or more vaccine compositions may also be provided. If a booster vaccination is performed, this booster vaccination will generally be between 1 week and 10 years, preferably 2 weeks and 6 years after the first administration of the composition to the individual (which in such cases is referred to as a "prime vaccination") administered to the same subject at a time between months.
  • a different vector such as one or more adenoviruses, or other vectors, such as modified Ankara's vaccinia virus (MVA) or DNA or protein, may also be administered to the subject after the priming vaccination.
  • a subject can be administered their recombinant viral vector as a prime and boosted with a composition comprising RSV F protein.
  • the administering comprises a priming administration and at least one booster administration.
  • administration is provided annually.
  • administration is provided annually with influenza vaccine.
  • compositions provided herein can be used with one or more other vaccines.
  • influenza vaccine Prevnar, tetanus vaccine, diphtheria vaccine, and pertussis vaccine.
  • vaccines provided herein may be used with any other vaccine indicated for use in pediatric patients.
  • the present application provides the pharmaceutical composition described above for use in inducing an immune response against RSV F protein.
  • % means wt%, that is, weight percentage. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional reagent products that can be purchased commercially.
  • the amino acid sequence 1-513 of the F protein was selected from the amino acid sequence shown in SEQ IN NO:1 (the corresponding nucleic acid sequence is the first position of the nucleic acid sequence of SEQ IN NO:2 -1539 position), at the C terminus of the amino acid sequence, T4 fibritin trimerization motif, thrombin site, 6x His-tag and Streptag II are sequentially fused from N terminus to C terminus.
  • the nucleic acid sequence encoding the above fusion sequence and plasmid pcDNA TM 3.1 (+) is constructed into recombinant plasmid pcDNA3.1-WT (SEQ ID NO:80), encoding and expressing RSV wild-type F (WT) protein extracellular domain fusion protein (SEQ ID NO:81).
  • Use point mutation method to mutate the nucleic acid sequence corresponding to the amino acid at the mutation site shown in Table 1 to obtain a recombinant plasmid containing the nucleic acid sequence encoding the mutant (SEQ ID NO: 35-62).
  • plasmid transfection doses were 0.3 ⁇ g/well and 1 ⁇ g/well.
  • the culture supernatant was collected, purified and identified according to conventional methods in this field to obtain mutants, which were numbered NeoC-RF-1-11, NeoC-RF-19-33 and NeoC-RF- 35-40, which are shown in Table 1, and the amino acid sequences are shown in SEQ ID NO: 3-34 respectively.
  • Example 2 ELISA detects the expression of the recombinant plasmid F protein and the expression of conformational epitopes in a mutant that combines an intramolecularly engineered disulfide bond mutation and a cavity-filling mutation.
  • This application utilizes monoclonal antibody D25 (D25 uses McLellan, JS, Chen, M., Leung, S., Graepel, KW, Du, X., Yang, Y., Zhou, T., Baxa, U., Yasuda, E.,Beaumont,T.,Kumar,A.,Modjarrad,K.,Zheng,Z.,Zhao,M.,Xia,N.,Kwong,PD,&Graham,BS(2013).Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science (New York, NY), 340 (6136), 1113–1117.
  • the recombinant plasmids were transfected into HEK293T cells cultured in 24-well plates as described in Example 1, and the plasmid transfection doses were 0.3 ⁇ g/well and 1 ⁇ g/well.
  • the cell culture supernatant was collected; 100 ⁇ l of the culture supernatant was added to Ni pre-coated ELISA plate wells (Thermo Scientific TM ) and incubated at room temperature for 2 hours, 250 ⁇ L PBST was added to each well and washed 3 times, and 100 ⁇ L/
  • the wells of monoclonal antibody D25 (3 ⁇ g/ml), or monoclonal antibody AM14 (3 ⁇ g/ml), or monoclonal antibody 4D7 (10 ⁇ g/ml) were incubated at room temperature for 2 hours, and 250 ⁇ L/well PBST was added to each well and washed 3 times, and 100 ⁇ L/well was added.
  • Figure 1B is a schematic diagram of the expression level of the pre-fusion conformation and trimerized F protein expressed by the recombinant plasmid of each mutant.
  • Figure 1C is a schematic diagram of the post-fusion conformation F protein expressed by the recombinant plasmid of each mutant. Schematic diagram of the expression level.
  • WT wild-type sequence protein
  • FIG. 1A Studies using the pre-fusion conformational F protein trimer-specific antibody AM14 show pre-fusion of recombinant plasmid-expressed NeoC-RF-35, NeoC-RF-37, NeoC-RF-39 and NeoC-RF-40 mutants.
  • the conformational, trimerized F protein is higher than that of the WT sequence protein, and the NeoC-RF-35 mutant is significantly higher than that of the WT sequence protein.
  • NeoC-RF-37, NeoC-RF-39 and NeoC-RF-40 the results are shown in Figure 1B.
  • Studies using the post-fusion conformation F protein-specific antibody 4D7 showed that NeoC-RF-39 and NeoC-RF-40 expressed significantly increased post-fusion conformation F protein, while NeoC-RF-35 hardly expressed post-fusion conformation F protein.
  • Fusion conformation of F protein see Figure 1C.
  • the stable trimer form of F protein in the pre-fusion conformation helps to further improve the ability of F protein to induce neutralizing antibodies. When the F protein conformation is unstable, it is easy to change from the pre-fusion conformation to the post-fusion conformation.
  • the F protein in the post-fusion conformation will greatly reduce or lose the ability to induce the production of neutralizing antibodies. It can be seen from the results in Figure 1A-1C that the NeoC-RF-35 mutant expresses significantly increased trimeric F protein with a stable pre-fusion conformation.
  • Example 3 ELISA detection of the expression level of pre-fuison conformation F protein expressed by mutant recombinant plasmid and determination of the stability of pre-fuison conformation F protein
  • the recombinant plasmids were transfected into HEK293T cells cultured in 24-well plates as described in Example 1, and the plasmid transfection doses were 0.3 ⁇ g/well and 1 ⁇ g/well.
  • the plasmid transfection doses were 0.3 ⁇ g/well and 1 ⁇ g/well.
  • collect the cell culture supernatant On the 3rd day after transfection, collect the cell culture supernatant; add 100 ⁇ l of the culture supernatant to the Ni pre-coated ELISA plate wells and incubate at room temperature for 2 hours. Then add 250 ⁇ L PBST to each well and wash 3 times. Add 100 ⁇ L/well pre-fuison conformation.
  • Antibody D25 monoclonal antibody D25 (10 ⁇ g/ml) was incubated at room temperature for 2 hours.
  • Figure 2A is a schematic diagram of the expression amount of the pre-fusion conformation F protein expressed by a mutant with a combination of engineered disulfide bond mutation and cavity filling
  • Figure 2B is a schematic diagram of the expression of the engineered disulfide bond mutation and cavity filling.
  • Figure 2C shows the engineered disulfide bond mutation, cavity filling, and amino acid substitution at position 215 with P. Schematic representation of the expression levels of F protein in the pre-fusion conformation of the combined mutants.
  • the mutants containing only one pair of engineered disulfide bonds (NeoC-RF-7, NeoC-RF-8, NeoC-RF-9) or only Contains two pairs of workers
  • the mutant with engineered disulfide bonds (NeoC-RF-10) cannot increase the expression of F protein in the pre-fusion conformation;
  • the mutant containing a pair of engineered disulfide bonds and at least one cavity filling combination (NeoC -RF-5, NeoC-RF-6, NeoC-RF-21, NeoC-RF-25, NeoC-RF-27, NeoC-RF-28, NeoC-RF-30 and NeoC-RF-35) were significantly expressed more High levels of pre-fusion conformation F protein;
  • the combination of the engineered disulfide bond mutation and cavity mutation of the present application, and/or combination with electrostatic mutation, and/or combination with mutation of amino acid 215 to proline can effectively improve pre -The expression level of F protein in fusion conformation.
  • the mutant transfected cell culture supernatant in Example 1 was stored at 4°C. On days 3, 10, 17, and 30 days after transfection, the amount of pre-fusion conformation F protein in the cell culture supernatant was detected by ELISA. , the results are shown in Figure 5. In the result analysis, the content of the pre-fusion conformation F protein expressed by each mutant on the 3rd day after transfection was set to 1, and the protein content of each mutant at other time points was expressed. It is the percentage of F protein content relative to the pre-fusion conformation on day 3.
  • the combination of engineered disulfide bond mutations and at least one cavity-filling mutation (NeoC-RF-5, NeoC-RF-6, NeoC-RF-30, NeoC-RF-35), engineered
  • a combination of engineered disulfide mutations, at least one cavity-filling mutation, and at least one electrostatic mutation (NeoC-RF-29) and engineered disulfide mutations, at least one cavity-filling mutation, and the amino acid at position 215 is proline
  • the stability of the pre-fusion conformation F protein expressed by the mutants with combinations of acid substitutions (NeoC-RF-19, NeoC-RF-20, NeoC-RF-36) was significantly increased, while only the cavity-filling mutations
  • the temporal stability of the F protein in the Pre-fusion conformation of the body (NeoC-RF-11) is significantly reduced.
  • Example 4 Expression map of neutralizing epitopes of pre-fusion conformation F protein expressed by recombinant plasmids encoding mutants
  • hRSV90 was prepared using Mousa, JJ, Kose, N., Matta, P., Gilchuk, P., & Crowe, JE, Jr (2017).
  • Figure 6A shows the pre-fusion expression of the recombinant plasmid encoding the mutant.
  • Neutralizing epitope of F protein in fusion conformation Site .
  • Figure 6B is a schematic expression map of the neutralizing epitope (Site IV and Site V) of the pre-fusion conformation F protein expressed by the recombinant plasmid encoding the mutant.
  • Figure 6C is the expression map of the recombinant plasmid encoding the mutant.
  • FIG. 6D Schematic diagram of the expression map of the neutralizing epitope (Site III) of the pre-fusion conformation F protein.
  • Figure 6D is the expression map of the neutralizing epitope (Site V) of the pre-fusion conformation F protein expressed by the recombinant plasmid encoding the mutant.
  • each neutralizing epitope of the pre-fusion conformation F protein expressed by the mutant recombinant plasmid is significantly higher than the expression of each neutralizing expression of the wild-type pre-fusion conformation F protein.
  • Example 5 Cells are transfected with mRNA encoding mutant F protein, and the F protein in pre-fusion conformation is expressed on the cell surface.
  • the nucleic acid sequence (SEQ IN NO:2) encoding the full-length sequence of RSV F protein (SEQ IN NO:1) was inserted between the BamHI and SacI sequences of the pNeoCura-Bvac plasmid vector through a gene synthesis protocol to obtain the recombinant plasmid pNeoCura- Bvac-WT is used as a plasmid template for preparing wild-type F protein mRNA.
  • the sequence of pNeoCura-Bvac-WT is shown in SEQ ID NO:68
  • its corresponding mRNA sequence (mRNA-NeoC-WT) is shown in SEQ ID NO :69 shown.
  • RNA Transcription Kit N1-Me-Pseudo UTP (Norvizan, DD4202) was used for in vitro transcription to obtain RNA.
  • RNA was purified using VAHTS RNA Clean Beads (Vazyme, N412-01).
  • RNA Cap 2'-O-Methyltransferase No. Weizan, DD4110 was used for capping reaction.
  • VAHTS RNA Clean Beads Vazyme, Cat. No.: N412-01 to purify the capped RNA to obtain the corresponding sequence of mRNA, namely SEQ ID: 63-67.
  • Cells were seeded in a 24-well plate (2.5 to 3 ⁇ 10 5 /well), cultured in DMEM medium (Thermo Fisher Scientific) containing 10% serum and 1% double antibody at 37°C, 5% CO 2 for 16-18 hours, and then transformed The cell density reached 80% confluency before staining.
  • DMEM medium Thermo Fisher Scientific
  • Opti-MEM medium Change to serum-free Opti-MEM medium (Thermo Fisher Scientific), mix mRNA (0.3 ⁇ g, 1 ⁇ g RNA/well) and Lipofectamine TM Messenger MAX TM transfection reagent (Thermo Fisher Scientific) to transfect HEK293T cells, and culture for 6 hours. Afterwards, the medium was replaced with DMEM medium (Thermo Fisher Scientific) containing 10% serum and 1% penicillin and streptomycin, and the culture was continued for 24 hours.
  • DMEM medium Thermo Fisher Scientific
  • Figure 8A is a schematic diagram of the average fluorescence intensity of the pre-fusion conformation F protein expressed on the cell surface combined with the antibody D25.
  • Figure 8B is a schematic diagram of the average fluorescence intensity of the pre-fusion conformation F protein expressed on the cell surface combined with the antibody AM14.
  • Example 6 Preparation of mRNA-LNP (lipid nanoparticles) of wild-type and mutant F proteins and use of mRNA-LNP to immunize BALB/c mice to induce serum antibodies and neutralizing antibodies
  • mice Female BALB/c mice aged 6-8 weeks (Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into 7 groups, with 5-10 mice in each group.
  • the negative control group was empty LNP without mRNA, and the immunization dose of wild-type and mutant F protein mRNA-LNP vaccines in each group was 10 ⁇ g/time/mouse.
  • Mice in each group were immunized by intramuscular injection on the 1st day (Prime) and the 21st day (Boost) of the experiment, and the immune volume was 50 ⁇ L.
  • the immune groups and doses are shown in Table 2.
  • the dose in each dose group is the dose of RNA.
  • mice in each experimental group On the 35th day of the experiment, blood was collected from mice in each experimental group through the retroorbital venous plexus, serum samples were separated, and inactivated by heating at 56°C for 30 minutes. The inactivated serum was diluted 2-fold with PBS buffer to obtain test serum samples. The ELISA method was used to detect antigen-specific antibodies in mouse serum, and the results are shown in Figure 9A. The results showed that mice immunized with each mRNA LNP induced the production of high-titer F protein-specific IgG, while the negative control group did not produce antibodies.
  • Day 1 Prepare HEp-2 cells (ATCC, Cat. No.: CCL-23) to ensure that the cell confluence is 90% on the second day;
  • mice immunized with the mRNA-LNP vaccine of the mutant F protein induced neutralizing antibody titers that were significantly higher than those of the mRNA-LNP vaccine with the wild-type F protein.
  • the mutant of the respiratory syncytial virus RSV F protein described in the embodiments of the present application has a more stable pre-fusion conformation and a higher expression level.
  • the expression level and stability of the trimerized pre-fusion protein are also significantly better than those of the wild-type F protein.
  • the mutants of the RSV F protein of respiratory syncytial virus are also significantly better than the wild-type F protein in the presentation of multiple neutralizing epitopes.
  • the mRNA vaccine expressing respiratory syncytial virus RSV mutant F protein significantly induced higher levels of neutralizing antibody titers by stabilizing the prefusion conformation and other neutralizing epitopes; therefore, this application
  • the F protein mutant is suitable for various vaccine forms with RSV F protein as the antigen, such as: mRNA vaccine, DNA vaccine, protein vaccine, adenovirus vaccine and recombinant virus particle vaccine.

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Abstract

La présente invention concerne un mutant de protéine F du VRS et son utilisation. La séquence d'acides aminés du mutant par rapport à la protéine F du VRS de type sauvage comprend au moins une mutation d'acide aminé. La mutation d'acide aminé est : 1) une combinaison d'au moins une mutation de liaison disulfure modifiée et d'au moins une mutation de remplissage de cavité ; ou 2) une combinaison d'au moins une mutation de liaison disulfure modifiée, d'au moins une mutation de remplissage de cavité, et d'au moins une mutation électrostatique, la mutation de remplissage de cavité comprenant la substitution d'un acide aminé à la position 190 avec F (la numérotation de la position étant basée sur une séquence telle que représentée dans SEQ ID NO : 1), et la mutation de liaison disulfure modifiée comprend la substitution d'acides aminés aux positions 466 et 443 avec C. Par comparaison à la protéine F du VRS de type sauvage, le mutant peut augmenter de manière significative le niveau d'expression de F ayant une conformation de pré-fusion, et a une stabilité accrue de la conformation de pré-fusion.
PCT/CN2023/091647 2022-04-29 2023-04-28 Mutant de protéine f du vrs et son utilisation WO2023208206A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117304280A (zh) * 2023-11-28 2023-12-29 江苏瑞科生物技术股份有限公司 一种重组rsv f蛋白及其应用
CN117586359A (zh) * 2024-01-19 2024-02-23 北京安百胜生物科技有限公司 一种具有免疫原性的呼吸道合胞病毒（rsv）多肽

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108738312A (zh) * 2015-12-23 2018-11-02 辉瑞公司 Rsv f蛋白突变体
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
WO2019032480A1 (fr) * 2017-08-07 2019-02-14 Avatar Medical, Llc Protéines f pré-fusion de rsv stabilisées en termes de conformation
WO2019147749A2 (fr) * 2018-01-29 2019-08-01 Merck Sharp & Dohme Corp. Protéines f rsv stabilisées et leurs utilisations
WO2021132379A1 (fr) * 2019-12-23 2021-07-01 田辺三菱製薬株式会社 Protéine f rsv mutante et son utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108738312A (zh) * 2015-12-23 2018-11-02 辉瑞公司 Rsv f蛋白突变体
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
WO2019032480A1 (fr) * 2017-08-07 2019-02-14 Avatar Medical, Llc Protéines f pré-fusion de rsv stabilisées en termes de conformation
WO2019147749A2 (fr) * 2018-01-29 2019-08-01 Merck Sharp & Dohme Corp. Protéines f rsv stabilisées et leurs utilisations
WO2021132379A1 (fr) * 2019-12-23 2021-07-01 田辺三菱製薬株式会社 Protéine f rsv mutante et son utilisation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117304280A (zh) * 2023-11-28 2023-12-29 江苏瑞科生物技术股份有限公司 一种重组rsv f蛋白及其应用
CN117304280B (zh) * 2023-11-28 2024-04-16 江苏瑞科生物技术股份有限公司 一种重组rsv f蛋白及其应用
CN117586359A (zh) * 2024-01-19 2024-02-23 北京安百胜生物科技有限公司 一种具有免疫原性的呼吸道合胞病毒（rsv）多肽

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