Stable pre-fusion RSV F protein and application thereof

Abstract

The invention discloses a stable pre-fusion RSV F protein and application thereof, the RSV F protein comprises one or more amino acid mutations to stabilize the RSV F protein in a pre-fusion conformation, and the RSV F protein has high affinity and neutralizing activity. Also disclosed are fusion proteins or multimers, immunogens, virus-like particles or protein nanoparticles comprising the RSV F protein, as well as nucleic acid molecules encoding the same, vectors, host cells, and immunogenic compositions.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a stable pre-fusion RSV F protein and an application thereof. Background Art

[0002] Human Respiratory Syncytial Virus (HRSV) is the most common pathogen causing viral pneumonia in children, which can cause interstitial pneumonia and bronchiolitis. RSV is a medium-sized single-stranded reverse-strand RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. RSV is similar to parainfluenza virus under an electron microscope. The virus particle size is about 150nm, which is slightly smaller than parainfluenza virus. It is sensitive to ether and has no hemagglutination. It forms unique syncytia in human epithelial tissue culture. The virus proliferates in the cytoplasm, and cytoplasmic inclusion bodies can be seen. Since respiratory syncytial virus is the main cause of bronchitis and pneumonia in infants and young children, it can also cause morbidity and death in immunodeficient patients and the elderly. Therefore, it is listed as one of the most prioritized vaccines in the world by the World Health Organization (WHO). The formalin-inactivated vaccine (FIRSV) developed in the 1960s not only failed to prevent infection in infants and young children, but also greatly increased the incidence and severity of bronchitis and pneumonia after vaccination. It is now believed that there are at least two reasons for this result: (1) FI-RSV failed to induce high-affinity, neutralizing antibodies, and failed to produce protective CD8+T cell responses, making the immunity induced by FI-RSV insufficient to control RSV infection (2) FI-RSV induced a Th2-biased CD4+T cell response, and the inflammatory response mediated led to enhanced disease (ERD). The low neutralizing antibody activity is mainly due to the lack of the prefusion F epitope on the surface of FI-RSV, and the Th2-biased immune response is one of the characteristics of formalin-inactivated virus vaccines. Therefore, a safe and effective RSV candidate vaccine should have both good neutralizing activity and induce a Th1-biased immune response.

[0003] There is no available vaccine against RSV infection, but the vaccine is highly needed due to the high burden of disease. RSV fusion glycoprotein (RSV F) is a striking vaccine antigen because it is the main target of antibodies in neutralizing human serum. Most of the neutralizing antibodies in human serum can resist the pre-fusion conformation, but due to its instability, the pre-fusion conformation has the tendency to be refolded into the post-fusion conformation in solution and on the surface of virions too early. Therefore, there is still a need for an effective vaccine against RSV, particularly a vaccine comprising the RSV F protein in the pre-fusion conformation. Summary of the invention

[0004] In order to develop RSV F protein with high expression level and stable pre-fusion conformation, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a stable pre-fusion RSV F protein, comprising one or more amino acid mutations, wherein the one or more amino acid mutations are present at positions 55, 100, 149, 189, 212, 254, 361, 362 and / or 458, or mutations corresponding to amino acids 55, 100, 149, 189, 212, 254, 361, 362 and / or 458 as determined by comparison with SEQ ID NO.1.

[0006] In some embodiments, the protein comprises a mutation at amino acid position 55, 189, 212, or 254.

[0007] In some embodiments, the protein comprises a mutation at amino acid position 55, 189, or 254.

[0008] In some embodiments, the protein comprises mutations at amino acids 55 and 189.

[0009] In some embodiments, the protein comprises mutations at amino acids 55 and 189, and mutations at amino acids 100, 149, 189, 212, 254, 361, 362 and / or 458.

[0010] In some embodiments, the protein comprises mutations at amino acids 55 and 189, and mutations at amino acids 100 and 361.

[0011] In some embodiments, the protein comprises mutations at amino acids 55 and 189, and mutations at amino acids 100, 254, and 361.

[0012] In some embodiments, the protein comprises mutations at amino acids 55 and 189, and mutations at amino acids 100, 362, and 212.

[0013] In some embodiments, the protein comprises mutations at amino acids 55 and 189, and mutations at amino acids 149 and 458.

[0014] In some embodiments, mutations at positions 55 and 189 are cavity-filling amino acid mutations; mutations at positions 100, 149, 361, 362, and 458 are amino acid mutations that introduce disulfide bonds; mutations at position 212 are amino acid mutations that introduce rotation-inducing polypeptide backbones; and mutations at position 254 are amino acid mutations that introduce chemical bonds.

[0015] In some embodiments, the cavity-filling amino acid mutation comprises a F, L, W, V, Y, H, or M substitution.

[0016] In some embodiments, the amino acid mutation that introduces a disulfide bond is a C substitution.

[0017] In some embodiments, the amino acid mutation that induces polypeptide backbone rotation is introduced as a P substitution.

[0018] In some embodiments, the amino acid mutation that introduces the chemical bond is an R substitution.

[0019] In some embodiments, the amino acid mutation at position 55, 100, 149, 189, 212, 254, 361, 362 or 458 is S55V, T100C, A149C, T189V, C212P, N254R, Q361C, S362C, Y458C, respectively.

[0020] In some embodiments, the RSV F protein further comprises a mutation at position 215, a furin cleavage site, or a deletion of an amino acid sequence comprising a furin cleavage site.

[0021] In some embodiments, the mutation at position 215 is S215P.

[0022] In some embodiments, the deletion of the furin cleavage site or the amino acid sequence comprising the furin cleavage site is a deletion of amino acids between 104-144.

[0023] In some embodiments, the deleted amino acid is replaced by a heterologous peptide linker.

[0024] In some embodiments, the linker is selected from G, S, GG, GS, SG, PS, GP, GGG, or GSG.

[0025] In a preferred embodiment, the linker is GS.

[0026] In some embodiments, the RSV F protein comprises the following amino acid mutations: S55V, T189V, S215P, and a deletion of amino acids between 104-144, wherein the deletion is replaced by a linker.

[0027] In a preferred embodiment, the RSV F protein comprises the following amino acid mutations: S55V, T189V, S215P, and a deletion of amino acids between 104-144, which is replaced by a linker GS.

[0028] In some embodiments, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, S215P, Q361C, and a deletion of amino acids between 104-144, wherein the deletion is replaced by a linker.

[0029] In a preferred embodiment, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, S215P, Q361C, and a deletion of amino acids between 104-144, which is replaced by a linker GS.

[0030] In some embodiments, the RSV F protein comprises the following amino acid mutations: S55V, A149C, T189V, S215P, Y458C, and a deletion of amino acids between 104-144, wherein the deletion is replaced by a linker.

[0031] In a preferred embodiment, the RSV F protein comprises the following amino acid mutations: S55V, A149C, T189V, S215P, Y458C, and a deletion of amino acids between 104-144, which is replaced by the linker GS.

[0032] In some embodiments, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, C212P, S362C, and a deletion of amino acids between 104-144, wherein the deletion is replaced by a linker.

[0033] In a preferred embodiment, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, C212P, S362C, and a deletion of amino acids between 104-144, which is replaced by a linker GS.

[0034] In some embodiments, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, N254R, Q361C, and a deletion of amino acids between 104-144, which is replaced by a linker.

[0035] In a preferred embodiment, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, N254R, Q361C, and a deletion of amino acids between 104-144, which is replaced by a linker GS.

[0036] In some embodiments, the RSV F protein is a full-length RSV F protein.

[0037] In some embodiments, the RSV F protein is a soluble RSV F protein.

[0038] In some embodiments, the soluble RSV F protein lacks the transmembrane region and the intracellular region.

[0039] The second aspect of the present invention provides a fusion protein or multimer comprising the RSV F protein described in the first aspect of the present invention.

[0040] In some embodiments, the fusion protein comprises a multimerization domain fused to the RSV F protein.

[0041] In some embodiments, the multimerization domain is a foldon domain.

[0042] In some embodiments, the multimer is formed by the association of RSV F fusion proteins by a multimerization domain.

[0043] In some embodiments, the multimer is a trimer.

[0044] In some embodiments, the fusion protein comprises a protease cleavage site.

[0045] In some embodiments, the protease is selected from thrombin, enterokinase, TEV protease, or 3C protease.

[0046] In some embodiments, the protease is selected from 3C protease.

[0047] In some embodiments, the fusion protein comprises a detection or purification tag.

[0048] In some embodiments, the tag is selected from a Strep tag, a Strep II tag, a FLAG tag, a glutathione S-transferase tag, a green fluorescent protein tag, a hemagglutinin A tag, a histidine tag, a luciferase tag, a maltose binding protein tag, a c-Myc tag, a protein A tag and / or a protein G tag.

[0049] In some embodiments, the tag is selected from a Strep II tag and a histidine tag.

[0050] In some embodiments, the peptides of the fusion protein, such as the F protein, the multimerization domain, the restriction site or the tag, are connected by a linker.

[0051] The third aspect of the present invention provides an immunogen comprising the RSV F protein described in the first aspect of the present invention or the fusion protein or multimer described in the second aspect of the present invention.

[0052] The fourth aspect of the present invention provides virus-like particles or protein nanoparticles, comprising the RSV F protein described in the first aspect of the present invention, the fusion protein or polymer described in the second aspect of the present invention, or the immunogen described in the third aspect of the present invention.

[0053] In some embodiments, the protein nanoparticles are ferritin nanoparticles, encapsulin nanoparticles, sulfur oxidoreductase (SOR) nanoparticles, 2,4-dioxotetrahydropterin synthase nanoparticles, or pyruvate dehydrogenase nanoparticles.

[0054] The fifth aspect of the present invention provides a nucleic acid molecule encoding the RSV F protein described in the first aspect of the present invention, the fusion protein or polymer described in the second aspect of the present invention, the immunogen described in the third aspect of the present invention, or the virus-like particle or protein nanoparticle described in the fourth aspect of the present invention.

[0055] In some embodiments, the nucleic acid molecule is codon optimized for expression in eukaryotic or prokaryotic cells.

[0056] In some embodiments, the nucleic acid molecule is operably linked to a promoter.

[0057] The sixth aspect of the present invention provides a vector comprising the nucleic acid molecule described in the fifth aspect of the present invention.

[0058] The seventh aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the fifth aspect of the present invention or the vector described in the sixth aspect of the present invention.

[0059] The eighth aspect of the present invention provides an immunogenic composition, comprising the RSV F protein described in the first aspect of the present invention, the fusion protein or polymer described in the second aspect of the present invention, the immunogen described in the third aspect of the present invention, the virus-like particle or protein nanoparticle described in the fourth aspect of the present invention, the nucleic acid molecule described in the fifth aspect of the present invention, the vector described in the sixth aspect of the present invention or the host cell described in the seventh aspect of the present invention; and a pharmaceutically acceptable carrier.

[0060] In some embodiments, the immunogenic composition further comprises an adjuvant.

[0061] In some embodiments, the adjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, and an oil emulsion adjuvant.

[0062] In some embodiments, the adjuvant comprises alum, AddaVax, MF59, AS01, AS03, AS04, MPL, QS21, a TLR9 agonist, a TLR7 agonist, a TLR4 agonist, a TLR3 agonist, or a combination of two or more thereof.

[0063] A ninth aspect of the present invention provides any of the following applications:

[0064] 1) Use of the RSV F protein described in the first aspect of the present invention, the fusion protein or polymer described in the second aspect of the present invention, the immunogen described in the third aspect of the present invention, the virus-like particle or protein nanoparticle described in the fourth aspect of the present invention, the nucleic acid molecule described in the fifth aspect of the present invention, the vector described in the sixth aspect of the present invention, the host cell described in the seventh aspect of the present invention, and the immunogenic composition described in the eighth aspect of the present invention in inhibiting / preventing RSV infection or in preparing products for treating / preventing diseases related to RSV infection;

[0065] 2) Use of the RSV F protein of the first aspect of the present invention, the fusion protein or polymer of the second aspect of the present invention, the immunogen of the third aspect of the present invention, the virus-like particle or protein nanoparticle of the fourth aspect of the present invention, the nucleic acid molecule of the fifth aspect of the present invention, the vector of the sixth aspect of the present invention, the host cell of the seventh aspect of the present invention, or the immunogenic composition of the eighth aspect of the present invention in the preparation of a product for inducing an immune response against RSV F protein in a subject or in the preparation of anti-RSV F protein antibodies;

[0066] 3) Use of the RSV F protein described in the first aspect of the present invention, the fusion protein or polymer described in the second aspect of the present invention, the immunogen described in the third aspect of the present invention, the virus-like particle or protein nanoparticle described in the fourth aspect of the present invention, the nucleic acid molecule described in the fifth aspect of the present invention, the vector described in the sixth aspect of the present invention, or the host cell described in the seventh aspect of the present invention in detecting RSV or preparing products for detecting diseases related to RSV infection.

[0067] The tenth aspect of the present invention provides any of the following methods:

[0068] 1) A method for detecting or isolating RSV F binding antibodies in a sample from a subject, comprising: contacting the RSV F protein described in the first aspect of the present invention, the fusion protein or multimer described in the second aspect of the present invention, the immunogen described in the third aspect of the present invention, the virus-like particle or protein nanoparticle described in the fourth aspect of the present invention with the RSV F binding antibody under the conditions of an immune complex; and

[0069] The immune complex is detected, thereby detecting or isolating the RSV F binding antibody in the subject.

[0070] In some embodiments, the subject is at risk for or has RSV infection.

[0071] In some embodiments, the RSV infection is a human RSV subtype A, a human RSV subtype B, or a bovine RSV infection.

[0072] In some embodiments, the subject is a human subject.

[0073] 2) A method for stabilizing the pre-fusion conformation of an RSV fusion polypeptide, introducing the mutation described in the first aspect of the invention.

[0074] 3) A method for preparing RSV F antibodies, using the RSV F protein described in the first aspect of the present invention, the fusion protein or polymer described in the second aspect of the present invention, and the immunogen described in the third aspect of the present invention to immunize non-human animals to obtain antibodies.

[0075] In some embodiments, the non-human animal is a vertebrate.

[0076] In some embodiments, the vertebrate comprises a mouse, a rat, a guinea pig, a rabbit, a sheep, or a non-human primate.

[0077] In some embodiments, the antibody comprises a chimeric, humanized antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is a graph showing the results of Western blot detection of RSV F protein in the expression supernatant.

[0079] Figure 2 is a sandwich ELISA test result diagram of RSV F protein in the expression supernatant; wherein, Figure 2 A is the ELISA test result of S63, S78 and S80 expression supernatants using D25 antibody; Figure 2 B is the result of ELISA test of S63, S78 and S80 expression supernatants using 4D7 antibody; Figure 2 C is the result of ELISA test of S105 and S110 expression supernatants using D25 antibody; Figure 2 D is the result of ELISA test of S105 and S110 expression supernatants using 4D7 antibody; Figure 2 E is the ELISA test result of the single point mutation expression supernatant using D25 antibody; Figure 2 F is the ELISA test result of the single point mutation expression supernatant using 4D7 antibody.

[0080] Figure 3 The figure is the sandwich ELISA test result of RSV F protein after heat shock at 50℃ in the expression supernatant; Figure 3 A is the ELISA test result of S63, S78 and S80 expression supernatants using D25 antibody; Figure 3 B is the result of ELISA test of S63, S78 and S80 expression supernatants using 4D7 antibody; Figure 3 C is the result of ELISA test of S105 and S110 expression supernatants using D25 antibody; Figure 3 D is the result of ELISA test of S105 and S110 expression supernatants using 4D7 antibody; Figure 3 E is the ELISA test result of the single point mutation expression supernatant using D25 antibody; Figure 3 F is the ELISA test result of the single point mutation expression supernatant using 4D7 antibody.

[0081] Figure 4 This is the result of SDS-PAGE electrophoresis.

[0082] Figure 5 This is a graph showing the titer of preF binding antibodies in serum induced by S63 detected by ELISA.

[0083] Figure 6 This is a graph showing the test results of neutralizing antibody titers in S63-induced serum.

[0084] Figure 7 This is a graph of preF binding antibody titers in serum induced by S80.

[0085] Figure 8 This is a graph showing the test results of neutralizing antibody titer in S80-induced serum.

[0086] Fig. 9 This is a sandwich ELISA test result of RSV (acession WPD93790.1) F protein in the expression supernatant; Fig. 9 A is a graph showing the results of ELISA using D25 antibody; Fig. 9 B is a graph showing the results of ELISA using 4D7 antibody; Fig. 9 C is a graph showing the ELISA test results of RSV F protein after heat shock at 50°C using D25 antibody; Fig. 9 D is a graph showing the ELISA detection results of RSV F protein after 50°C heat shock using 4D7 antibody.

[0087] Fig.10 This is a sandwich ELISA test result of RSV Beijing10-04 strain F protein in the expression supernatant; Fig.10 A is a graph showing the results of ELISA using D25 antibody; Fig.10 B is a graph showing the results of ELISA using 4D7 antibody; Fig.10 C is a graph showing the ELISA test results of RSV F protein after heat shock at 50°C using D25 antibody; Fig.10 D is a graph showing the ELISA detection results of RSV F protein after 50°C heat shock using 4D7 antibody.

[0088] Fig.11These are the results of the mouse immune detection of the F protein of type A RSV (acession WPD93790.1), wherein 11A is the preF binding antibody titer in the serum, 11B is the ELISA endpoint dilution result, and 11C is the detection result of the neutralizing antibody titer in the serum. DETAILED DESCRIPTION OF THE INVENTION

[0090] The present invention has been extensively and thoroughly studied, and by mutating different amino acids, some new mutant RSV F proteins with improved properties have been found. As shown in the examples of the present invention, these new mutant RSV F molecules have a preF conformation with a higher expression level, and a high immunogenicity.

[0091] In the present invention, the use of alternatives (such as "or") should be understood to refer to any one, all or any combination of the alternatives. The terms "include", "have" and "include" are used synonymously in the present invention, and these terms and their variations are intended to be interpreted as non-restrictive.

[0092] In the present invention, the term "RSV F protein" includes all forms of the protein, including F1 polypeptide and F2 polypeptide, i.e., including F0 precursor polypeptide, trimer of F0 precursor polypeptide, RSV F protomer and mature RSV F trimer - whether membrane-bound or soluble. As used in the present invention, the term "mutated RSV F protein" refers to an RSV F protein containing one or more artificially introduced / artificial mutations.

[0093] In the present invention, the terms "protein" and "polypeptide" are used interchangeably.

[0094] In the present invention, the terms "nucleic acid molecule", "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" are used interchangeably.

[0095] In the present invention, the term "adjuvant" refers to a substance that can enhance, accelerate or prolong the body's immune response to an immunogen or immunogenic composition (such as a vaccine).

[0096] RSV F protein

[0097] RSV is known to exist as a single serotype with two antigenic subtypes A and B. The amino acid sequences of the two types of mature processed F proteins are approximately 93% identical. As in the present invention, the amino acid position is given with reference to the RSV F protein sequence (AC083301.1, SEQ ID NO.1) from the A2 strain. Therefore, the term "amino acid at position "x" of the RSV F protein" means the amino acid corresponding to the amino acid at position "x" in the RSV F protein of the RSVA2 strain of SEQ ID NO.1. Note that in the numbering system used in this application, 1 refers to the N-terminal amino acid (SEQ ID NO.1) of an immature F0 protein. When a RSV strain is used instead of the A2 strain, the amino acid position of the F protein will be numbered by inserting spaces where necessary to align the sequences of other RSV strains with the F protein of SEQ ID NO.1, with reference to the numbering of the F protein of the A2 strain of SEQ ID NO.1. Sequence alignment is performed using methods well known in the art, for example, by CLUSTALW, Bioedit or CLC Workbench.

[0098] The mutation in the present invention can be introduced into any suitable RSV F sequence to make it in the preF conformation. Suitable RSV F sequences are sequences of WT / natural RSV F molecules. Suitable RSV F sequences also include sequences of mutant RSV F molecules, i.e., sequences comprising one or more artificially introduced mutations compared to WT / natural RSV F molecules.

[0099] Those skilled in the art will appreciate that proteins can be mutated by conventional molecular biology procedures. The pre-fusion RSV F polypeptide in the composition according to the present invention is stable, i.e., it is not easy to change into the post-fusion conformation when processing the polypeptide (e.g., such as purification, freeze-thaw cycles and / or storage, etc.).

[0100] In the present invention, RSV F protein is derived from RSV subtype A or RSV subtype B. RSV subtype A includes but is not limited to RSV A2 strain, RSV Long strain, RSV S2 strain, RSV Line 19 strain, hRSV / A / Germany / 22-01307 / 2021 (ACCESSIONWPD93790) strain, wherein RSV A2 strain is preferred. RSV subtype B includes but is not limited to RSV 18537 strain, RSV B1 strain, RSV 9320 strain, RSV Beijing 10-04 (ACCESSIONAPW77892) strain.

[0101] In certain embodiments, the RSV F protein is derived from the RSVA2 virus strain of SEQ ID NO.1. The amino acid sequence of SEQ ID NO.1 is the amino acid sequence of the F0 polypeptide of the F protein of the RSVA2 strain, and the various components of the F0 polypeptide are described with reference to this. The amino acid sequence at positions 1-25 is a signal sequence, also known as a leader sequence or signal peptide; the amino acid sequence at positions 26-109 is the F2 polypeptide; the amino acid sequence at positions 110-136 is the pep27 region; the amino acid sequence at positions 137-513 is the extracellular region of the F1 polypeptide; the amino acid sequence at positions 514-574 is the transmembrane region and the intracellular region of the F1 polypeptide. It should be noted that the amino acid sequence of the F1 polypeptide (transmembrane region and intracellular region) can be a sequence that has more than 85%, 90% or 95% identity with the amino acid sequence at positions 514-574 of sequence number 1.

[0102] In some embodiments, the RSV F protein does not comprise a signal sequence.

[0103] In the present invention, embodiments of the specific exemplary amino acid sequences of the aforementioned mutations, or embodiments of the specific regions (e.g., F1 and / or F2 regions) of these sequences, variant forms equivalent to these amino acid sequences can also be used. In some embodiments, it is possible to use an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the RSV F amino acid sequence of the present invention, across its F1 and F2 regions (especially across the 26th-109th (F2) and the 137th-513th (part of F1 that does not include transmembrane and cytoplasmic domains) amino acid residues). Compared with one or more specific amino acid sequences provided by the invention, these variant forms may have added, deleted or replaced amino acids. Therefore, their length may be longer or shorter than a specific sequence.

[0104] Similarly, the amino acid residues after position 514 of each exemplary RSV F amino acid sequence provided by the present invention can be removed or changed. For example, in several amino acid sequences provided by the present invention, the amino acid residues after position 514 include natural RSV F transmembrane and cytoplasmic domains. In some embodiments, the transmembrane and cytoplasmic domains of these natural RSV F can be removed to produce a soluble (i.e., non-membrane-bound) version of the RSV F molecule, or can be replaced with different transmembrane and / or cytoplasmic domains.

[0105] The mutant RSV F protein can be obtained and / or isolated and / or maintained in the preF conformation using any suitable method known in the art, including but not limited to standard protein purification methods, such as ion exchange chromatography, size exclusion chromatography, and / or affinity chromatography.

[0106] In some embodiments, a mutant RSV F in a preF conformation can be obtained, isolated, or maintained by controlling the ionic strength of the medium / buffer in which the protein is present (e.g., by using a medium of high or low ionic strength). In some embodiments, a mutant RSV F protein can be obtained, isolated, or maintained at one or more temperatures that are conducive to preserving the preF conformation. In some embodiments, a mutant RSV F protein can be obtained, isolated, or maintained over a period of time that reduces the extent of loss of the preF conformation. The extent to which a protein formulation maintains one or more desired conformations (e.g., a preF conformation) can be determined by any suitable method known in the art, including but not limited to biochemical, biophysical, immunological, and virological analyses. These assays include, but are not limited to, for example, immunoprecipitation, ELISA or ELISA spot assays, crystallography (including co-crystallization with antibodies), sedimentation, analytical ultracentrifugation, dynamic light scattering (DLS), electron microscopy (EM), cryo-EM tomography, calorimetry, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), circular dichroism analysis, small angle x-ray scattering, neutralization assays, antibody-dependent cellular cytotoxicity assays, and / or in vivo challenge studies.

[0107] Fusion Protein

[0108] One embodiment of the present invention is a fusion protein comprising the aforementioned mutant RSV F protein and a multimerization domain.

[0109] The multimerization domain is not particularly limited as long as it is a polypeptide required for multimerization of the mutant RSV F protein, and examples thereof include a foldon domain for trimerization.

[0110] As the Foldon domain, the Foldon domain derived from the minor fiber protein (fibritin) of T4 phage can be exemplified.

[0111] In a specific embodiment of the present invention, the sequence of the folding subdomain is shown as SEQ ID NO.2.

[0112] The fusion protein also comprises a protease cleavage site, and the protease includes but is not limited to thrombin, enterokinase, TEV protease or 3C protease.

[0113] In one embodiment, the protease is selected from 3C protease.

[0114] In a specific embodiment of the present invention, the sequence of the protease cleavage site is shown as SEQ ID NO.3.

[0115] Fusion protein can include the polypeptide except mutant RSV F protein and multimerization domain, and the polypeptide except this mutant RSV F protein and multimerization domain is not particularly limited as long as it does not hinder the effect of aforementioned mutant RSV F protein. The label of detection or purification RSV F molecule can be illustrated, including but not limited to Strep label, Strep II label, FLAG label, glutathione S-transferase (GST) label, green fluorescent protein (GFP) label, hemagglutinin A (HA) label, histidine (His) label, luciferase label, maltose binding protein (MBP) label, c-Myc label, protein A label or protein G label.

[0116] In some embodiments of the invention, the fusion protein comprises a Strep II tag and a histidine tag.

[0117] The peptides of the fusion protein of the present invention are connected by a linker. The linker can be composed of 1 to 50 consecutive amino acids, 1 to 30 consecutive amino acids, or 1 to 15 amino acids. As an optional embodiment, exemplary linkers include but are not limited to SAIG, GRS, G, SA.

[0118] Virus-like particles

[0119] In some embodiments, provide and comprise the virus-like particle (VLP) of the mutant RSV F albumen as mentioned above that is stabilized in the pre-fusion conformation.VLP lacks the virus component required for viral replication and therefore presents the virus of highly attenuated form.When being applied to the experimenter, VLP can present the polypeptide (for example, being stabilized in the recombinant RSVF albumen of the pre-fusion conformation) that can induce the immune response for RSV.Virus-like particle and its manufacture method are known and well-known to those of ordinary skill in the art, and the known formation VLP from the viral protein of some viruses, including human papilloma virus, HIV, human polyoma virus, rotavirus, parvovirus, canine parvovirus, hepatitis E virus and Newcastle disease virus.For example, chimeric VLP containing RSV antigen and can be VLP based on Newcastle disease virus.VLP based on Newcastle disease has previously been shown to induce the neutralization immune response for RSV in mice.The formation of these VLPs can be detected by any suitable technology. Examples of techniques known in the art that are suitable for detecting VLPs in culture medium include, for example, electron microscopy, dynamic light scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction and / or size exclusion chromatography of VLPs), and density gradient centrifugation.

[0120] Protein Nanoparticles

[0121] In some embodiments, a protein nanoparticle is provided that includes one or more of any of the disclosed mutant RSV F proteins stabilized in a prefusion conformation, wherein the protein nanoparticle is specifically bound by a prefusion-specific antibody (e.g., D25 or CR9501 antibody), and / or includes a RSV F prefusion-specific conformation (e.g., antigenic site Or V). Non-limiting examples of nanoparticles include ferritin nanoparticles, encapsulin nanoparticles, and sulfur oxidoreductase (SOR) nanoparticles, which are composed of assemblies of monomeric subunits including ferritin, encapsulin protein, and SOR protein, respectively. To construct protein nanoparticles including the disclosed recombinant RSV F protein stabilized in a pre-fusion conformation, an antigen is linked to a subunit of a protein nanoparticle (e.g., ferritin, encapsulin protein, or SOR protein). The fusion protein self-assembles into nanoparticles under appropriate conditions.

[0122] The RSV F protein included on the protein nanoparticle can be a human subtype A, human subtype B, or bovine RSV F protein that includes the substitutions disclosed herein for prefusion stabilization.

[0123] Nucleic acid molecules

[0124] In some embodiments, the present invention provides nucleic acid molecules encoding mutant RSV F proteins of the present invention, and vectors comprising the nucleic acid molecules. In view of the generally known and understood nature of the genetic code among those of ordinary skill in the art, those of ordinary skill in the art can easily determine the nucleic acid sequence of the nucleic acid molecule encoding any mutant RSV F protein of the present invention.

[0125] Any suitable method known in the art can be used to obtain or prepare nucleic acid molecules encoding mutant RSV F molecules of the present invention. For example, nucleic acid molecules encoding mutant RSV F molecules can be obtained from cloned DNA, or prepared by chemical synthesis. In some embodiments, nucleic acid molecules can be obtained by reverse transcription RNA, wherein RNA is prepared by any method known to those of ordinary skill in the art. Point mutations, or any other modifications described in the present invention (for example, removing C- terminal sequences, replacing C- terminal sequences, etc.), can be completed by DNA standard recombination methods known and understood by those of ordinary skill in the art.

[0126] In certain embodiments, the nucleic acid molecules encoding the protein according to the present invention are codon optimized to express in eukaryotic cells or prokaryotic cells, preferably in mammalian cells, more preferably in human cells. If at least one non-preferred codon is replaced by a more preferred codon as compared to the wild-type sequence, the sequence is considered to be codon optimized. Here, a non-preferred codon is a codon that is not as frequently used as another codon encoding the same amino acid in an organism, and a more preferred codon is a codon that is more frequently used than a non-preferred codon in an organism. Preferably more than one non-preferred codon, preferably the most or all non-preferred codons, are replaced by more preferred codons. Preferably, the codon most frequently used in an organism is used for a codon optimized sequence. Replacement by preferred codons generally results in higher expression.

[0127] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acid molecules can encode the same polypeptide. It should also be understood that the skilled person can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect the codon usage of any specific host organism to be expressed polypeptide. Therefore, unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may or may not include introns.

[0128] Carrier

[0129] One embodiment of the present invention is a vector comprising a nucleic acid molecule encoding the aforementioned RSV F protein. As vectors, plasmid vectors and viral vectors can be exemplified, and vectors capable of being expressed in prokaryotic cells, vectors capable of being expressed in eukaryotic cells, and vectors capable of being expressed in cells derived from mammals can be cited. For example, bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from a combination of plasmids and phage DNA; viral DNA such as vaccinia, adenovirus, fowlpox virus, pseudorabies, adenovirus, adeno-associated virus, retrovirus, etc. The vector is an expression vector suitable for generating a polypeptide expression product. In the expression vector, the nucleic acid encoding the PreF antigen is usually arranged in proximity and orientation to a suitable transcription control sequence (promoter, and optionally, one or more enhancers) to direct mRNA synthesis. Examples of such promoters include: the immediate early promoter of CMV, LTR or SV40 promoter, the polyhedrin promoter of baculovirus, the lac or trp promoter of Escherichia coli, phage T7 and promoters, and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also typically contains a ribosome binding site and a transcription terminator for translation initiation. Optionally, the vector comprises a sequence suitable for amplified expression. In addition, optionally, the expression vector comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as kanamycin, tetracycline or ampicillin resistance in Escherichia coli.

[0130] Host cells

[0131] Another embodiment of the present invention is a host cell, which comprises the aforementioned nucleic acid molecule or vector. As a host cell, as long as it can express the protein expressed by the aforementioned nucleic acid molecule, it is not particularly limited, and can be a host cell used for standard genetic engineering technology. Specifically, non-limiting examples of suitable host cells can be exemplified, including bacteria, archaea, insects, fungi (e.g., yeast), plants and animal cells (e.g., mammalian cells, such as humans). Exemplary cells used include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian bone marrow and lymphocyte lines. The proliferation technology of mammalian cells in culture is well known. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK and COS cell lines, but the following cell lines can be used, such as cells designed to provide higher expression, desired glycosylation patterns or other characteristics. In some embodiments, the host cell includes HEK293 cells or derivatives thereof, such as GnTI - / - cell.

[0132] Host cells can be transformed with recombinant DNA by conventional techniques as are well known to those skilled in the art. When the host is prokaryotic (e.g., but not limited to E. coli), competent cells capable of DNA uptake can be prepared from cells collected after the exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed by electroporation after the protoplasts of the host cells are formed or when necessary.

[0133] When the host is a eukaryotic organism, these DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of plasmids encapsulated in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding a disclosed antigen and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector such as Simian Virus 40 (SV40) or bovine papilloma virus to transiently infect or transform eukaryotic cells and express the protein.

[0134] By culturing host cells under suitable conditions, the aforementioned mutant RSV F protein or fusion protein can be expressed to produce the aforementioned mutant RSV F protein, fusion protein or their polymer. The aforementioned mutant RSV F protein, fusion protein or their polymer produced can be purified using known means.

[0135] Composition

[0136] The mutated RSVF protein, virus-like particle, protein nanoparticle, nucleic acid molecule, host cell disclosed in the present invention and optional adjuvant or optionally other therapeutic ingredients such as antiviral drugs are combined together. In some embodiments, the composition including one or more disclosed RSV F proteins, virus-like particles, protein nanoparticles, nucleic acid molecules or host cells is an immunogenic composition. The composition may include any RSVF protein, including a recombinant RSVF protein as disclosed in the present invention (e.g., a protein nanoparticle including any recombinant RSVF protein as disclosed in the present invention), including any virus-like particle of a recombinant RSVF protein as disclosed in the present invention, encoding any nucleic acid molecule of a recombinant RSVF protein as disclosed in the present invention, or encoding or including any vector of a recombinant RSVF protein as disclosed in the present invention.

[0137] The immunogenic composition may include an adjuvant. An adjuvant is a vehicle for enhancing antigenicity. In the present application, adjuvants include, but are not limited to, suspensions of minerals (alum, aluminum hydroxide or phosphate) adsorbed with antigens; or water-in-oil emulsions, for example, wherein the antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including inactivated mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibit antigen degradation and / or cause macrophage influx). Immunostimulatory oligonucleotides (such as those including CpG motifs) can also be used as adjuvants. Adjuvants include biomolecules ("biological adjuvants"), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and toll-like receptor (TLR) agonists, such as TLR-9 agonists, TLR7 agonists, TLR4 agonists, TLR3 agonists.

[0138] In some embodiments, the adjuvant comprises one or more of aluminum salt adjuvant, surfactant, polynucleotide, lipopolysaccharide, liposome, and oil emulsion adjuvant. For example, Alum, AddaVax, CpG, Alum+CpG, MF59, AS04, AS03, AS01, AS01E, MPL, QS21, etc. MF59 is a water-mixed adjuvant, mainly composed of three parts: oil phase, emulsifier and adjuvant; the oil phase is a mixture of micronized short-chain triglycerides suitable for human injection; the emulsifier is a surfactant that can make the oil phase and water evenly mixed; the adjuvant mainly includes glycerol, threitol, ATP and other moisturizers and buffers. AS04 adjuvant is a mixture of AS03 adjuvant and MPL adjuvant. AS03 adjuvant is a mixture of liposome, TWEEN80, and SORBITAN three surfactants. AS01E is a nano-liposome solution prepared from DOPC, Chol, MPL and QS-21. The main component of MPL adjuvant is lipopolysaccharide.

[0139] These compositions can be administered to a subject by a variety of modes of administration known to those of ordinary skill in the art, for example, nasally, pulmonary, intramuscularly, subcutaneously, intravenously, intraperitoneally, or parenterally.

[0140] In order to prepare the composition, the disclosed RSV F protein, fusion protein or polymer, immunogen, virus-like particle, protein nanoparticle, carrier or nucleic acid molecule can be combined with various pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, for example, excipients, lubricants, binders and disintegrants in solid preparations or solvents, cosolvents, suspending agents, isotonic agents, buffers and analgesics in liquid preparations. It is also possible to further appropriately and appropriately use additives such as common preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents as needed.

[0141] Examples of dosage forms for parenteral administration include injection preparations, external preparations, suppositories for inhalation, ophthalmic preparations, eye ointments, nasal drops, ear drops, liposome preparations, and the like.

[0142] The dosage form for oral administration can be exemplified by solid or liquid dosage forms, specifically, tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, lozenges and the like.

[0143] The composition of the present invention is preferably in the form of a liquid, preferably an injection.

[0144] Pharmaceutical compositions are normally sterile and stable under conditions of manufacture, storage and use. Can be by the disclosed RSV F albumen, virus-like particle, protein nanoparticle, viral vector or nucleic acid molecule of required amount be incorporated in having suitable solvent of the present invention, then filter sterilization when necessary to prepare sterile solution.Usually, dispersion is prepared by the disclosed antigen and / or other bioactivator being incorporated in the sterile vehicle containing basic dispersion medium and other required components from those listed by the present invention.In the case of sterile powder, preparation method comprises vacuum drying and freeze drying, and it obtains the disclosed antigen plus the powder of any additional required components from its previous aseptic filtered solution.Can realize the prevention of microbial action by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid etc.The actual method for preparing the composition that can be applied will be known or obvious to those skilled in the art.

[0145] In some embodiments, the composition includes an adjuvant. Adjuvants are well known to those of ordinary skill in the art, for example, those that can be included in immunogenic compositions. In some embodiments, the adjuvant is selected to induce a Th1 biased immune response in a subject administered an immunogenic composition, the immunogenic composition comprising an adjuvant and a disclosed antigen, or a nucleic acid or viral vector encoding, expressing or including the antigen.

[0146] The amount of disclosed RSV F protein, viral vector or nucleic acid molecule can be changed according to, for example, the specific antigen used, route of administration and scheme and target population. The amount utilized in the immunogenic composition is selected based on a subject population (e.g., infants or the elderly). The optimal amount of a particular composition can be determined by standard studies involving antibody titers and other reactions observed in a subject. It should be understood that the antigen of the therapeutically effective amount in the immunogenic composition can include an amount that cannot induce an immune response by administering a single dose, but is effective (e.g., in a primary immunity-boosting administration scheme) after administering multiple doses.

[0147] In several examples, pharmaceutical compositions for inducing an immune response to RSV in humans include a disclosed RSV F protein, viral vector, or nucleic acid molecule for administration to an infant (e.g., an initial dose age of birth to 1 year old, e.g., an infant of 0 to 6 months) or an elderly patient subject (e.g., a subject older than 65 years old) in a therapeutically effective amount. It should be understood that the choice of adjuvant may be different in these different applications, and those skilled in the art can empirically determine the optimal adjuvant and concentration for each situation.

[0148] Treatment

[0149] In some embodiments, the disclosed RSV F protein, or nucleic acids encoding, expressing, or including RSV F protein, fusion proteins or polymers, immunogens, vectors, virus-like particles, or protein nanoparticles are used in a subject to induce an immune response against RSV. Thus, in some embodiments, a therapeutically effective amount of an immunogenic composition comprising one or more of the disclosed RSV F protein, or nucleic acids encoding, expressing, or including the antigen, or viral vectors can be administered to a subject to generate an immune response against RSV.

[0150] In the present invention, a preventive or therapeutically effective amount of an immunogenic composition comprising RSV F protein, or a nucleic acid, vector, virus-like particle or protein nanoparticle encoding, expressing or comprising the antigen is administered to a subject in need of treatment for a duration and conditions sufficient to prevent, inhibit and / or improve RSV infection in the subject. The immunogenic composition is administered in an amount sufficient to induce an immune response to RSV antigens such as RSV F protein in the subject.

[0151] In some embodiments, selection is for example due to being exposed to RSV or being exposed to the possibility of RSV and having RSV infection or being in the subject under the risk of suffering from RSV infection for treatment.After applying the disclosed therapeutic composition of therapeutically effective amount, RSV infection, the symptom relevant to RSV infection or both can be monitored to the subject.Because almost all mankind can infect RSV before 3 years old, include whole birth population as immunization related group.This can be for example by any time from birth to 6 months old, from 6 months old to 5 years old, in pregnant women (or women of childbearing age) to protect the family members of their baby, newborn or fetus and age greater than 50 years old subject by the passive transfer of antibody and start immunization scheme to carry out.

[0152] Immunogenic compositions can be used in collaborative vaccination schemes or combined preparations. In certain embodiments, the combined immunogenic compositions and collaborative immunization schemes use independent immunogens or preparations, each for inducing an immune response to RSV antigens, such as an immune response to RSV F protein. The independent immunogenic compositions inducing an immune response to RSV antigens can be combined in a multivalent immunogenic composition administered to a subject in a single immunization step, or they can be administered independently in a collaborative immunization scheme (in a monovalent immunogenic composition).

[0153] The administration of immunogenic compositions may be for preventive or therapeutic purposes. When provided prophylactically, the immunogenic compositions are provided before any symptoms, for example, before infection. The prophylactic administration of immunogenic compositions is used to prevent or improve any subsequent infection. When provided therapeutically, the immunogenic compositions are provided during or after the onset of symptoms of disease or infection, for example, after the development of symptoms of RSV infection, or after RSV infection diagnosis. Therefore, immunogenic compositions can be provided before expected exposure to RSV to reduce the expected severity, duration or degree of infection and / or related disease symptoms, or after exposure or suspected exposure to the virus, or after infection actual initiation.

[0154] For preventive and therapeutic purposes, the immunogenic composition can be delivered by a single bolus, and in an extended period of time, it can be delivered continuously (for example, continuously transdermally, mucosally or intravenously), or administered to a subject with a repeated administration scheme (for example, by repeated administration schemes every hour, every day or once a week). The therapeutically effective dose of the immunogenic composition can be provided with repeated doses in an extended prevention or treatment scheme, and the scheme will produce clinically significant results to alleviate one or more symptoms or detectable symptoms related to the target disease or condition as described in the present invention. The determination of effective dose in the present invention is generally based on animal model research, followed by human clinical trials, and is guided by the administration scheme of the occurrence or severity of the target disease symptoms or condition in the subject that is significantly reduced. Suitable models in this regard include, for example, rodents, rats, pigs, cats, ferrets, non-human primates, and other recognized animal model subjects as known in the art. Alternatively, in vitro models (for example, immunology and histopathological assays) can be used to determine effective doses. Using these models, only routine calculations and adjustments are required to determine appropriate concentrations and dosages to administer a therapeutically effective amount of an immunogenic composition (e.g., an amount effective to elicit a desired immune response or to alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of an immunogenic composition may simply inhibit or enhance one or more selected biological activities associated with a disease or condition as described herein for therapeutic or diagnostic purposes.

[0155] In one embodiment, suitable immunization scheme comprises at least three independent inoculations with one or more immunogenic compositions, wherein the second inoculation is greater than about two weeks, about three weeks to eight weeks or about four weeks after the first inoculation.Usually, the third inoculation is used in several months after the second inoculation, and in a specific embodiment, greater than about five months after the first inoculation, greater than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation.Also need to exceed the regular inoculation outside the third time to strengthen the "immune memory" of the experimenter.Can be by obtaining the aliquot of serum from the experimenter and measuring antibody titer in the immunization procedure process to determine the appropriateness of selected vaccination parameters, for example, formula, dosage, scheme etc.If this detection shows, vaccination is suboptimal, then the experimenter can strengthen with the immunogenic composition of additional dose, and can expect to regulate the vaccination parameters in the mode of enhancing immune response.Expectation can have several strengthenings, and each strengthening can include the same or different RSV F albumen.

[0156] For the initial immunization-boosting scheme, the initial immunization can be administered in a single dose or multiple doses, for example, two doses, three doses, four doses, five doses or six doses or more can be administered to the subject in a few days, weeks or months. Reinforcement can be administered in a single dose or multiple doses, for example, two to six doses or more can be administered to the subject in a day, a week or months. Multiple reinforcements can also be given, for example, once to five times or more. Different dosages can be used in a series of continuous inoculations. For example, a relatively large dose is used in the primary inoculation, and then a relatively small dose is reinforced. An immune response to the selected antigen surface can be generated by inoculating the subject once or multiple times with the immunogenic composition disclosed in the present invention.

[0157] Application of mutant RSV F

[0158] In some embodiments, the mutated RSV F of the present invention can be used as a research tool, a diagnostic tool, a therapeutic agent, for the production of antibody reagents or as a target for therapeutic antibodies, and / or a component of a vaccine or vaccine composition. The vaccine includes but is not limited to subunit vaccines such as recombinant vaccines, virus-like particle vaccines, etc., viral vector vaccines, nucleic acid vaccines such as mRNA vaccines, DNA vaccines; for example, in some embodiments, the mutated RSV F of the present invention can be used as a vaccine immunogen for animal subjects (e.g., mammalian subjects, including humans). It should be understood by those skilled in the art that the mutated RSV F of the present invention can also be used in various other applications, and all of these applications and uses are intended to fall within the scope of the present invention.

[0159] Studying Anti-RSV F Antibodies

[0160] In one embodiment, the mutated RSV F proteins of the present invention can be used as an analyte for testing and / or measuring the binding and / or titer of anti-RSV F antibodies, for example in an ELISA assay, a Biacore / SPR binding assay, and / or any other antibody binding assay known in the art. For example, the mutated RSV F proteins of the present invention can be used to analyze and / or compare the effects of anti-RSV F antibodies.

[0161] Producing antibodies

[0162] The mutant RSV F protein of the present invention or a material comprising a mutant RSV F protein can also be used to produce therapeutic antibodies, and / or antibodies that can be used as research tools, or for any other desired purposes. For example, the mutant RSV F protein of the present invention can be used for immunization to obtain RSV F protein antibodies used as research tools and / or therapeutic agents. In some embodiments, in order to produce antibodies, the mutant RSV F protein of the present invention can be used to immunize non-human animals, such as vertebrates, including but not limited to mice, rats, guinea pigs, rabbits, goats, non-human primates, etc. Such antibodies can then be obtained from animals, which can be monoclonal antibodies or polyclonal antibodies, and / or cells that produce such antibodies. For example, in some embodiments, the mutant RSV F protein of the present invention can be used to immunize mice to produce and obtain monoclonal antibodies, and / or hybridomas that produce such monoclonal antibodies. These methods can be implemented using standard methods known in the art for producing mouse monoclonal antibodies, including standard methods for producing hybridomas. In some embodiments, the mutant RSV F proteins of the invention can be used to generate chimeric (e.g., partially human), humanized, or fully human antibodies, for example, using any method currently known in the art for generating chimeric, humanized, and fully human antibodies, including but not limited to CDR grafting methods, phage display methods, transgenic mouse methods (e.g., using mice genetically modified to be able to produce fully human antibodies, such as transgenic mice), and / or any other suitable method known in the art. Antibodies against mutant RSV F proteins of the invention prepared using such a system can be characterized for antigenicity using one or a group of multiple antigens (preferably including the mutant RSV F proteins of the invention themselves). Such antibodies can be additionally characterized by any standard method known to those of ordinary skill in the art, including, but not limited to, ELISA-based methods, SPR-based methods, biochemical methods (e.g., but not limited to isoelectric point determination), and methods known in the art for studying the biodistribution, safety, and efficacy of antibodies (e.g., in preclinical and clinical studies).

[0163] Subjects

[0164] In the present invention, the subject includes any animal species, especially those susceptible to RSV infection or can provide a model animal system for RSV infection research. In some embodiments, the subject is a mammalian species. In some embodiments, the subject is an avian species. Mammalian subjects include, but are not limited to humans, non-human primates, rodents, rabbits and ferrets. Avian subjects include, but are not limited to chickens, such as chickens in poultry farms. In a preferred embodiment, the subject is a human.

[0165] product

[0166] The present invention also provides products comprising mutant RSV F proteins of the present invention, or compositions comprising the mutant RSV F proteins. For ease of use of the methods and compositions of the present invention, any component and / or composition of the present invention, as well as other components for experiment or treatment or vaccination purposes, can be packaged in the form of a test kit. Typically, in addition to the above-mentioned components, the test kit also includes other materials, which may include, for example, instructions for use of the components, packaging materials, containers and / or delivery devices.

[0167] The label or package insert will also typically include instructions for use of RSV F protein, or nucleic acid or vector encoding, expressing or including the antigen, for example, in a method for treating or preventing RSV infection. Package inserts typically include instructions routinely included in the commercial packaging of the therapeutic product, which contain information about indications, usage, dosage, administration, contraindications and / or warnings about the use of such therapeutic products. The explanatory material can be written in electronic form (e.g., a computer floppy disk or CD) or can be visual (e.g., a video file). The kit may also include other components to facilitate specific applications designed for the kit. The kit may additionally include buffers and other reagents commonly used to implement ad hoc methods. These kits and appropriate inclusions are well known to those skilled in the art. DETAILED DESCRIPTION

[0168] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0169] Example 1F Protein modification and screening

[0170] 1.1F protein modification and plasmid construction

[0171] The F protein of the reference sequence RSV A2 strain (GeneBank AC083301.1, SEQ ID NO.1) was used as a template and was modified as follows: the extracellular region at positions 1-513 was truncated, and the trimerization domain Foldon (SEQ ID NO.2), 3C restriction site (SEQ ID NO.3), His tag (SEQ ID NO.4) and strep tag (SEQ ID NO.5) were fused to its C-terminus; a connecting sequence was added between each tag; the sequence of the F protein terminal fusion protein is shown in SEQ ID NO.6.

[0172] The sequence of RSV A2 strain F protein (GeneBank AC083301.1) was codon optimized; and the base sequence corresponding to amino acids 1-513 was cloned into the pcDNA3.1 vector, namely pcDNA-wtSF.

[0173] According to the structure of preF, several point mutations were designed in the extracellular region to obtain an F protein that is stable in the pre conformation. The point mutations involved include: 1. The amino acids 104-144 are deleted in the extracellular region, and a glycine-serine dipeptide linker (sc9-10) is added between positions 103 and 145; 2. The amino acid at position 215 is mutated from serine to proline (i.e. S215P); 3. The amino acid at position 55 is mutated from serine to valine (i.e. S55V); 4. The amino acid at position 189 is mutated from threonine to valine (i.e. T189V); 5. The amino acid at position 100 is mutated from valine to cysteine ​​(i.e. T10 0C); 6. the 361st amino acid mutated from glutamine to cysteine ​​(i.e., Q361C); 7. the 254th amino acid mutated from asparagine to arginine (i.e., N254R); the 149th amino acid mutated from alanine to cysteine ​​(i.e., A149C); the 458th amino acid mutated from tyrosine to cysteine ​​(i.e., Y458C); the 212th amino acid mutated from cysteine ​​to proline (i.e., C212P); the 362nd amino acid mutated from serine to cysteine ​​(i.e., S362C).

[0174] A total of multiple mutants were designed for this mutation, namely S55V, T189V, C212P, N254R, S63, S80, S78, S105 and S110, among which S63 contained mutations sc9-10, S215P, S55V, T189V; S80 contained mutations sc9-10, S215P, S55V, T189V, T100C, Q361C; S78 contained mutations sc9-10, S215P, S55V, T189V, A149C, Y458C; S105 contained mutations sc9-10, T189V, S55V, T100C, S362C, C212P; S110 contained mutations sc9-10, S55V, T189V, N254R, T100C, Q361C.

[0175] Using pcDNA-wtSF as a template, primers overlapping with the vector sequence were designed at the 5' and 3' ends of the wtSF expression frame; forward and reverse primers overlapping with each other were designed at the point mutation and deletion positions. The mutation was integrated into the recombinant plasmid using homologous recombination. The correct clones were identified by gene sequencing for plasmid extraction, and the DNA concentration was detected by spectrophotometer and frozen in a -80℃ refrigerator for storage. At the same time, sequencing was performed again to identify the correctness of the plasmid sequence.

[0176] 1.2293F cell transfection and supernatant detection

[0177] The mutant plasmid and the control plasmid were co-transfected into 293F cells, and the expression supernatant was harvested to detect and analyze the expression and conformation of the F protein in the supernatant. The transfected plasmids included S55V, T189V, C212P, N254R, S63, S80, S78, S105, S110, wt (wild type), post (deletion of positions 137-146 of the F protein; Jason S.McLellan et al., 2011), DS-Cav1 (preF protein modification of the vaccine approved by GSK, including mutations S155C, S190F, V207L, S290C; Jiang Qian et al., 2018.), 847 (preF protein modification of the vaccine approved by Pfizer, including mutations T103C, I148C, S190V, D486S, Ye Che et al., 2023).

[0178] 1.2.1 Transfection of 293F cells

[0179] (1) When 293F grows to 2.5×10 6 / ml, dispense into disposable filter membrane centrifuge tubes at a volume of 10ml / tube.

[0180] (2) Take a 1.5 ml centrifuge tube, add 500 μl of blank culture medium, then add 10 μg of plasmid, and mix well; in another 1.5 ml centrifuge tube, add 450 μl of blank culture medium, then add 40 μl of PEI, and mix well.

[0181] (3) Mix the solutions in the two centrifuge tubes, let them stand at room temperature for 15 min, then add them dropwise to the 293F cells and culture them in a shaker.

[0182] (4) After 4 days of culture, the supernatant was collected by centrifugation at 3000 rpm for 15 min.

[0183] 1.2.2 Western blot detection of F protein expression in supernatant

[0184] (1) Mix the expression supernatant with loading buffer, add β-mercaptoethanol to the reducing buffer and omit β-mercaptoethanol to the non-reducing buffer, and heat at 95°C for 5 minutes.

[0185] (2) Add the cooked sample to 4%-12% SDS-PAGE gel and perform electrophoresis at 120V for 70 minutes.

[0186] (3) After electrophoresis, the protein was transferred to the PVDF membrane using the wet transfer method.

[0187] (4) Add 5% skim milk in PBS to block for 1 hour.

[0188] (5) After washing with PBST, the PVDF membrane was placed in PBST containing human Motavizumab antibody (10,000-fold dilution) and incubated at 37°C for 1 hour.

[0189] (6) After washing three times with PBST, the PVDF membrane was placed in PBST containing the secondary antibody Anti-human IgG HRP (2500-fold dilution) and incubated at 37°C for 1 hour.

[0190] (7) Wash the membrane three times with PBST. While washing the membrane for the last time, prepare fresh luminescent working solution. Pick up the membrane with tweezers, drain the PBST, fix it in a dark box, and add luminescent solution. Place the membrane in a multifunctional chemiluminescent imager for photographing and development.

[0191] (8) Results

[0192] Western blot results are as follows Figure 1 As shown, it shows that S63, S80, S78, S105 and S110 can express proteins, and the expression levels are higher than those of wt, 847 and DS-Cav1 in the control group. In addition, the results of non-reducing gel showed that S78, S80, S105 and S110 with interchain disulfide bonds (T100C and Q361C, T100C / S362C, A149C / Y458C) formed disulfide bonds between the monomers of the trimer.

[0193] 1.2.3 Sandwich ELISA to detect the conformation of F protein in the supernatant

[0194] (1) Coat the ELISA plate with Anti-Strep antibody overnight.

[0195] (2) The next day, add blocking solution and block for 1 hour.

[0196] (3) Discard the solution, add the culture supernatant of each F protein, and incubate for 1 hour.

[0197] (4) After discarding the liquid, wash three times, add serially diluted F protein antibodies D25 (McLellan JS et al., 2013) and 4D7 (Jessica A. et al., 2016), and incubate for 1 hour.

[0198] (5) After discarding the solution, wash three times, add HPR-labeled Anti-human IgG secondary antibody, and incubate for 45 minutes.

[0199] (6) After discarding the liquid, wash three times and add a color developing substrate for color development.

[0200] The antibody information used is as follows:

[0201]

[0202] (7) Results

[0203] The results of sandwich ELISA are as follows Figure 2 As shown, S55V, T189V, N254R, S63, S78, S80, S105 and S110 have a stronger ability to bind to the pre conformation-specific antibody D25, while C212P, N254R, S63, S78, S80, S105 and S110 have a weaker ability to bind to the post conformation-specific antibody 4D7, indicating that the above mutations can promote the F protein to be in the pre conformation.

[0204] 1.3 Thermal stability analysis

[0205] The supernatant obtained in 1.2.1 was placed in a 50°C water bath for heat shock. After 1 hour of heat shock, the ability of the protein to bind to D25 and 4D7 antibodies was detected according to the sandwich ELISA method in 1.2.3 to evaluate the thermal stability of the protein.

[0206] The results are as follows Figure 3 As shown, the sandwich ELISA results after the expression supernatant was heat shocked at 50°C for 1 hour showed that S55V, N254R, S63, S78, S80, S105 and S110 still had strong D25 binding ability and maintained weak 4D7 binding ability, indicating that the above mutants have good thermal stability.

[0207] 2 Protein expression and purification

[0208] The above study evaluated the F protein in the expression supernatant, and the results showed that S63 and S80 were expressed at high levels, had obvious pre conformations and good thermal stability. The next step was to express and purify the protein and further evaluate its affinity with the antibody at the level of purified protein.

[0209] 2. Transfection of 1293F

[0210] Plasmids of S63, S80, wt, post, DS-Cav1, and 847 were extracted and sequenced to verify the accuracy of the sequences. 1L 293F cells were transfected according to the method in 1.2.1, and the supernatant was harvested 4 days after transfection.

[0211] 2.2 Protein purification

[0212] 2.2.1Strep magnetic bead affinity purification and gel filtration chromatography

[0213] (1) After filtering the supernatant using a 0.45 μm glass fiber filter, the supernatant was concentrated using a tangential flow system and the buffer was replaced with PBS.

[0214] (2) Add Strep magnetic beads to the protein solution in the concentration and replacement buffer, and then incubate the bottle on a shaker at 4°C overnight.

[0215] (3) Clean the outer surface of the magnetic rod sleeve with ultrapure water and wipe it dry with absorbent paper; place the magnetic rod sleeve into the bottle after incubation of protein and magnetic beads, insert the magnetic end of the magnetic rod into the sleeve, and gently stir in the bottle to absorb more magnetic beads; remove the sleeve with the magnetic rod and insert it into a 50mL centrifuge tube; remove the magnetic rod and rinse the magnetic beads adsorbed on the outer surface of the sleeve with the balancing solution.

[0216] (4) Repeat step (3) to fully recover the magnetic beads in the bottle.

[0217] (5) Insert the 50 mL centrifuge tube containing the collected magnetic beads into the magnetic rack and gently turn it upside down to allow the magnetic beads to be fully adsorbed onto the magnetic stickers of the magnetic rack. Unscrew the tube cap, press the tube rack, and slowly pour out the equilibration solution from the side opposite to the magnetic beads adsorption side.

[0218] (6) Add balancing solution and wash the magnetic beads three times. After the last wash solution is discarded, remove the remaining balancing solution and aspirate.

[0219] (7) Remove the centrifuge tube, add 10 mL of eluent and let stand for 10 minutes. Use a magnetic stand to adsorb the magnetic beads, aspirate the eluent and transfer it to a new centrifuge tube, and measure the protein concentration using a spectrophotometer.

[0220] (8) Use an ultrafiltration concentrator to concentrate the protein solution. Add the protein solution to a pre-equilibrated ultrafiltration concentrator and centrifuge at 4000×g to concentrate to 1 ml. Aspirate the concentrate and measure the protein concentration using a spectrophotometer.

[0221] (9) After affinity purification of the F protein using strep magnetic beads, the protein was further purified using a gel filtration column Superdex200 Increase, and the protein solution corresponding to the F protein was collected.

[0222] 2.2.2SDS-PAGE

[0223] The protein separated and purified by gel filtration chromatography was analyzed by SDS-PAGE electrophoresis. Figure 4 As shown, the furin cleavage site of wt, post, 847, and DS-Cav1 was not mutated, and the resulting proteins were cleaved into F1 and F2; S63 and S80 were mutated and the furin cleavage site was missing, resulting in uncleaved, single-chain F protein.

[0224] Analysis of 3F protein physicochemical properties

[0225] After obtaining the purified F protein, the protein particle size and thermal stability Tm value were analyzed using the UNCLE instrument. The results are shown in the following table. The particle size distribution of S63 and S80 is uniform, with the Tm value of S63 being 59.32 and the Tm value of S80 being 73.89.

[0226]

[0227]

[0228] 4F protein and antibody affinity determination

[0229] The affinity between F protein and antibody was determined using surface plasmon resonance technology. The antibody information involved is as follows:

[0230]

[0231] (1) Covalent coupling of protein A on the CM5 chip.

[0232] (2) Antibody D25 is flowed into the chip and captured.

[0233] (3) After the antibody is captured, DS-Cav1 protein is injected into the cell from low concentration to high concentration.

[0234] (4) After detecting proteins of various concentrations, a pH 2.0 glycine solution was added to regenerate the chip.

[0235] (5) Detect S63, S80 and post proteins respectively according to the methods of (2) to (4).

[0236] (6) According to the methods of (1) to (5), CR9501 (Anders Krarup et al., 2015), MPE8 (Davide Corti et al., 2013), AM14 (Morgan SAGilman et al., 2015), and 4D7 antibodies were captured respectively, and the affinity between DS-Cav1, S63, S80 and post proteins and each antibody was detected in turn.

[0237] (7) Results

[0238] The results are shown in the table below. The results of SPR detection of the affinity between F protein and antibodies showed that S63 and S80 had higher affinity with pre-conformation antibodies (D25, CR9501, AM14, MPE8), and lower affinity with post-conformation antibodies (4D7).

[0239]

[0240] 5 Mouse immunization experiment

[0241] 5.1S63 mouse immunization experiment

[0242] 2 μg of protein post, DS-Cav1, and S63 were mixed with AddaVax adjuvant and then immunized into SPF BALB / c mice of about 7 weeks old. A PBS immunization control group was set up at the same time. A booster immunization was performed two weeks after the first immunization. The immunization method was intramuscular injection. Blood was collected 3 weeks after the second immunization, and the serum was separated and inactivated to detect the titer of preF-specific antibodies and neutralizing antibodies in the serum.

[0243] 5.1.1 Detection of preF-specific antibody titer in serum

[0244] (1) DS2 protein (M Gordon Joyce et al., 2016) was coated onto an ELISA plate at 50 ng per well overnight.

[0245] (2) The next day, add blocking solution and block for 1 hour.

[0246] (3) Discard the solution, add gradient diluted mouse serum, and incubate for 1 hour.

[0247] (4) After discarding the solution, wash three times, add HPR-labeled Anti-mouse IgG secondary antibody, and incubate for 45 minutes.

[0248] (5) After discarding the liquid, wash three times and add a color developing substrate for color development.

[0249] (6) Results

[0250] The results of ELISA are as follows Figure 5 As shown, S63 induced higher levels of preF binding antibody titers.

[0251] 5.1.2 Determination of neutralizing antibody titer in serum

[0252] (1) Dilute RSV virus solution to 200 TCID 50 .

[0253] (2) Serum was serially diluted in a 96-well microplate. The first well was diluted 20-fold, followed by 3-fold dilutions for a total of 6 dilutions, with 5 replicate wells for each dilution.

[0254] (3) Add 50 μl of the diluted virus solution to each well, mix well, and incubate in a 37°C 5% CO2 incubator for 60 min. At the same time, set up a virus control group and a normal cell control group.

[0255] (4) After incubation, add 100 μl of HEp2 cell suspension to each well and place in a 37°C 5% CO2 incubator for culture.

[0256] (5) Observe and record the results daily for 5-7 days.

[0257] (6) According to the Reed-Muench method, the serum dilution factor when the cell half-well lesion occurs is calculated, i.e., PD 50 .

[0258] (7) Results

[0259] The results of serum neutralizing antibody titer test are as follows Figure 6 As shown, S63 was shown to induce higher neutralizing antibody titers.

[0260] 5.2S80 mouse immunization experiment

[0261] 10 μg of protein post, DS-Cav1, and S80 were mixed with AddaVax adjuvant and then immunized into SPF BALB / c mice of about 7 weeks old. Two weeks after the first immunization, booster immunization was performed. Immunization was by intramuscular injection. Blood was collected 3 weeks after the second immunization, and the serum was separated and inactivated to detect the titer of preF-specific antibodies and neutralizing antibodies in the serum.

[0262] 5.2.1 Detection of preF-specific antibody titers in serum

[0263] Detect the titer of preF-specific antibodies in the collected serum according to the method in 5.1.1.

[0264] Test results such as Figure 7 As shown, S80 induced higher levels of preF binding antibody titers.

[0265] 5.2.2 Determination of neutralizing antibody titers in serum

[0266] Detect RSV neutralizing antibody titer in the collected serum according to the method in 5.1.2.

[0267] The results of the test are as follows Figure 8 As shown, S80 induced higher neutralizing antibody titers.

[0268] Example 2 Functional study of F protein mutations in different virus strains

[0269] The F protein of type A virus strain hRSV / A / Germany / 22-01307 / 2021 (ACCESSION WPD93790) and type B virus strain Beijing10-04 (ACCESSION APW77892) were used as reference sequences for mutant design, and the conformation of the F protein was further detected by sandwich ELISA, the physicochemical properties of the purified F protein, the affinity of the F protein and the pre-conformation-specific antibody were analyzed, and the titer of specific antibodies and the titer of neutralizing antibodies in the serum were detected by mouse immunization experiments. The method is the same as in Example 1. The results of sandwich ELISA for detecting the conformation of the F protein are shown in Figure 1. Figure 9-10 As shown, for different virus strains, the mutations designed in the present application can promote the F protein to be in the pre conformation and have good thermal stability.

[0270] The Tm values ​​and particle sizes of different modified clones of the F protein with hRSV / A / Germany / 22-01307 / 2021 (ACCESSION WPD93790) as the reference sequence were analyzed using the UNCLE instrument. The results are shown in the table below. S78, S80, and S110 have higher Tm values, and the particle size distribution is around 13-14 nm.

[0271] sample S78 S80 S110 Tm(℃) 71.86 74.06 82.91 Particle size 14.26 13.16 13.2

[0272] The affinity of the F protein with the type A virus strain hRSV / A / Germany / 22-01307 / 2021 (ACCESSION WPD93790) as the reference sequence and the pre conformation-specific antibodies are shown in the following table. S78, S80, and S110 have higher affinity with the pre conformation-specific antibodies D25, CR9501, MPE8, and AM14.

[0273]

[0274]

[0275] Mouse immunoassay results Fig.11 As shown, the F protein mutant with type A virus strain hRSV / A / Germany / 22-01307 / 2021 (ACCESSION WPD93790) as the reference sequence can produce higher levels of binding antibodies and neutralizing antibodies.

[0276] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

[0277] References:

[0278] Jason S. McLellan et.al., Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes. 2011.

[0279] Jiang Qian et.al., Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein. 2018.

[0280] Ye Che et.al., Rational Design of a Highly Immunogenic Prefusion-Stabilized F Glycoprotein Antigen for a Respiratory Syncytial Virus Vaccine. 2023.

[0281] McLellan JS et.al., Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science. 2013.

[0282] Jessica A. Flynn et.al., Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein. 2016.

[0283] Anders Krarup et.al.A highly stable prefusion RSV F vaccinederivedfrom structuralanalysis ofthe fusion mechanism.2015.

[0284] Davide Corti et.al.,Cross-neutralization of four paramyxoviruses by ahumanmonoclonal antibody.2013.

[0285] Morgan S.A.Gilman et.al.,Characterization of a Prefusion-SpecificAntibody ThatRecognizes a Quaternary,Cleavage-Dependent Epitope on the RSVFusionGlycoprotein.2015.

[0286] M Gordon Joyce et.al.,Iterative structure-based improvement ofafusion-glycoprotein vaccine against RSV.2016.

Claims

1. A stable pre-fusion RSV F protein, characterized in that The RSV F protein comprises one or more amino acid mutations, wherein the one or more amino acid mutations are present at positions 55, 100, 149, 189, 212, 254, 361, 362 and / or 458, or mutations corresponding to amino acids 55, 100, 149, 189, 212, 254, 361, 362 and / or 458 as determined by alignment with SEQ ID NO.1; Preferably, the protein comprises a mutation in amino acid positions 55, 189, 212 and / or 254; Preferably, the protein comprises a mutation at amino acid position 55, 189 or 254; Preferably, the protein comprises mutations at amino acids 55 and 189; Preferably, the protein further comprises mutations in amino acids 100, 149, 189, 212, 254, 361, 362 and / or 458; Preferably, the protein further comprises mutations at amino acids 100 and 361; Preferably, the protein further comprises mutations at amino acids 100, 254, and 361; Preferably, the protein further comprises mutations at amino acids 100, 362, and 212; Preferably, the protein further comprises mutations at amino acids 149 and 458; Preferably, mutations at positions 55 and 189 are amino acid mutations that fill the cavity; mutations at positions 100, 149, 361, 362, and 458 are amino acid mutations that introduce disulfide bonds; mutations at position 212 are amino acid mutations that introduce rotation-inducing polypeptide backbones; and mutations at position 254 are amino acid mutations that introduce chemical bonds. Preferably, the cavity-filling amino acid mutation comprises a F, L, W, V, Y, H or M substitution; Preferably, the amino acid mutation that introduces a disulfide bond is a substitution of C; Preferably, the amino acid mutation that induces rotation of the polypeptide backbone is introduced as a substitution of P; Preferably, the amino acid mutation that introduces the chemical bond is a substitution of R; Preferably, the amino acid mutation at position 55, 100, 149, 189, 212, 254, 361, 362 or 458 is S55V, T100C, A149C, T189V, C212P, N254R, Q361C, S362C, Y458C, respectively; Preferably, the RSV F protein further comprises a mutation at position 215, and / or a deletion of a furin cleavage site or an amino acid sequence comprising a furin cleavage site; Preferably, the mutation at position 215 is S215P; Preferably, the deletion of the furin cleavage site or the amino acid sequence comprising the furin cleavage site is a deletion of amino acids between 104 and 144; Preferably, the deleted amino acid is replaced by a heterologous peptide linker; Preferably, the linker is selected from G, S, GG, GS, SG, PS, GP, GGG or GSG; Preferably, the linker is GS; Preferably, the RSV F protein comprises the following amino acid mutations: S55V, T189V, S215P, amino acid deletion between 104-144, the deletion being replaced by the linker preferably GS; Preferably, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, S215P, Q361C, amino acid deletion between 104-144, the deletion being replaced by the linker preferably GS; Preferably, the RSV F protein comprises the following amino acid mutations: S55V, A149C, T189V, S215P, Y458C, amino acid deletion between 104-144, the deletion being replaced by the linker preferably GS; Preferably, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, C212P, S362C, amino acid deletions between 104-144, the deletions being replaced by the linker preferably GS; Preferably, the RSV F protein comprises the following amino acid mutations: S55V, T100C, T189V, N254R, Q361C, amino acid deletion between 104-144, the deletion being replaced by the linker preferably GS; Preferably, the RSV F protein is a full-length RSV F protein; Preferably, the RSV F protein is a soluble RSV F protein; Preferably, the soluble RSV F protein lacks the transmembrane region and the intracellular region.

2. A fusion protein or polymer, characterized in that: Comprising the RSV F protein of claim 1; Preferably, the fusion protein comprises a multimerization domain fused to the RSV F protein; Preferably, the multimerization domain is a foldon domain; Preferably, the multimer is formed by the association of the multimerization domains of the RSV F fusion protein; Preferably, the polymer is a trimer; Preferably, the fusion protein comprises a protease cleavage site; Preferably, the protease is selected from thrombin, enterokinase, TEV protease or 3C protease; Preferably, the protease is selected from 3C protease; Preferably, the fusion protein comprises a detection or purification tag; Preferably, the tag is selected from the group consisting of a Strep tag, a Strep II tag, a FLAG tag, a glutathione S-transferase tag, a green fluorescent protein tag, a hemagglutinin A tag, a histidine tag, a luciferase tag, a maltose binding protein tag, a c-Myc tag, a protein A tag and / or a protein G tag; Preferably, the tag is selected from the group consisting of a Strep II tag and a histidine tag; Preferably, the peptides of the fusion protein are connected via a linker.

3. An immunogen, characterized in that Comprising the RSV F protein of claim 1, the fusion protein or multimer of claim 2.

4. Virus-like particles or protein nanoparticles, characterized in that Comprising the RSV F protein of claim 1, the fusion protein or polymer of claim 2, and the immunogen of claim 3; Preferably, the protein nanoparticles are ferritin nanoparticles, encapsulin nanoparticles, sulfur oxidoreductase (SOR) nanoparticles, 2,4-dioxotetrahydropterin synthase nanoparticles or pyruvate dehydrogenase nanoparticles.

5. A nucleic acid molecule, characterized in that Encoding the RSV F protein of claim 1, the fusion protein or multimer of claim 2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4; Preferably, the nucleic acid molecule is codon optimized for expression in eukaryotic or prokaryotic cells; Preferably, the nucleic acid molecule is operably linked to a promoter.

6. A carrier, characterized in that Comprising the nucleic acid molecule of claim 5.

7. A host cell, characterized in that Comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. An immunogenic composition, characterized in that Comprising the RSV F protein of claim 1, the fusion protein or polymer of claim 2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6 or the host cell of claim 7; and a pharmaceutically acceptable carrier; Preferably, the immunogenic composition further comprises an adjuvant; Preferably, the adjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, and an oil emulsion adjuvant; Preferably, the adjuvant comprises alum, AddaVax, MF59, AS01, AS01E, AS03, AS04, MPL, QS21, a TLR9 agonist, a TLR7 agonist, a TLR4 agonist, a TLR3 agonist or a combination of two or more thereof.

9. Any of the following applications: 1) Use of the RSV F protein of claim 1, the fusion protein or polymer of claim 2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7, and the immunogenic composition of claim 8 in inhibiting / preventing RSV infection or in preparing products for treating / preventing RSV infection-related diseases; 2) Use of the RSV F protein of claim 1, the fusion protein or polymer of claim 2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7, and the immunogenic composition of claim 8 in the preparation of a product for inducing an immune response against RSV F protein in a subject or in the preparation of anti-RSV F protein antibodies; 3) Use of the RSV F protein of claim 1, the fusion protein or polymer of claim 2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6 or the host cell of claim 7 in detecting RSV or preparing products for detecting RSV infection-related diseases.

10. Either of the following methods: 1) A method for detecting or isolating RSV F binding antibodies in a sample from a subject, comprising: contacting under conditions of using the RSV F protein of claim 1, the fusion protein or multimer of claim 2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4 and the immune complex between the RSV F binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV F binding antibody in the subject; Preferably, the subject is at risk for or has RSV infection; Preferably, the RSV infection is human RSV subtype A, human RSV subtype B or bovine RSV infection; Preferably, the subject is a human subject; 2) A method for stabilizing the pre-fusion conformation of an RSV fusion polypeptide, characterized by introducing the mutation described in claim 1; 3) A method for preparing RSV F antibodies, characterized in that the antibodies are obtained by immunizing non-human animals using the RSV F protein of claim 1, the fusion protein or polymer of claim 2, and the immunogen of claim 3; Preferably, the non-human animal is a vertebrate; Preferably, the vertebrates include mice, rats, guinea pigs, rabbits, sheep, and non-human primates; Preferably, the antibody comprises a chimeric or humanized antibody.

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