Respiratory syncytial virus f protein mutant and use thereof

Abstract

Provided are an RSV F protein mutant, a nucleic acid molecule or a vector encoding the RSV F protein mutant, an immunogenic composition comprising the RSV F protein mutant, the nucleic acid molecule, or the vector, and use thereof. The RSV F mutant can bind, with high affinity, to a preF-specific neutralizing antibody, but it basically does not bind to an antibody that specifically binds to a postF protein.

Description

Respiratory syncytial virus F protein mutants and their uses

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202510124781.3, filed on January 26, 2025, the contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to respiratory syncytial virus (RSV) F protein mutants and their uses, and more specifically, to RSV F protein mutants that bind with high affinity to RSV preF protein-specific neutralizing antibodies and substantially do not bind to RSV postF protein-specific antibodies. Background Technology

[0004] Respiratory syncytial virus (RSV) is a seasonal, highly contagious pathogen. The U-shaped age curve of RSV infection in humans shows that the peak incidence occurs in infants under 5 years of age and the elderly over 65 years of age. Most patients with RSV infection experience only mild symptoms and recover spontaneously within 1-2 weeks. More severe infections, if left untreated, can develop into lower respiratory tract infections (LRTIs) with more serious clinical manifestations and may further progress to chronic respiratory and lung diseases. Globally, RSV causes approximately 3 million hospitalizations in children under 5 years of age and approximately 60,000 in-hospital deaths annually. Treatment for high-risk groups such as children and the elderly primarily involves symptomatic and supportive care and symptom relief. Natural RSV infection does not induce a long-term immune memory response against conserved immunogens; RSV immunization requires passive prophylaxis (antibody drugs) or active prophylaxis (prophylactic vaccines).

[0005] RSV belongs to the genus Orthopneumovirus of the family Pneumoviridae. It is an enveloped, non-segmented, negative-sense RNA virus. The RSV genome is approximately 15–16 kb in size and encodes 11 proteins, including 8 structural proteins and 3 non-structural proteins (NS1, NS2, and M2-2). The structural proteins include 3 transmembrane surface proteins (G, F, and SH), 2 matrix proteins (M and M2-1), and 3 nucleocapsid proteins (L, N, and P). RSV surface proteins are the main targets of neutralizing antibodies and potential vaccine antigens. There are two RSV subtypes, A and B, which differ mainly in the G glycoprotein, while the F glycoprotein sequence is more conserved between the two subtypes.

[0006] Mature F glycoprotein has three domains: extracellular domain (ECD), transmembrane domain (TM), and cytoplasmic tail (CT). The cytoplasmic tail contains a single palmitoylated cysteine ​​residue.

[0007] The human RSV F glycoprotein is first translated from mRNA into a single 574-amino acid polypeptide precursor (called "F0" or "F0 precursor") containing an N-terminal signal peptide sequence (amino acids 1-25). After translation, the signal peptide is removed in the endoplasmic reticulum by a signal peptidase. The remaining portion of the F0 precursor (i.e., residues 26-574) can be further cleaved at two sites (109 / 110 and 136 / 137) by cellular proteases (especially furin proteases), removing a 27-amino acid intercalation sequence named pep27 (or p27) (amino acids 110-136) to produce two fragments named F2 (N-terminal portion; amino acids 26-109) and F1 (C-terminal portion; amino acids 137-574). F1 contains its N-terminal hydrophobic fusion peptide (FP) and two heptapeptide repeat regions (HRA and HRB). HRA is located near FP, and HRB is located near the TM domain. F1 and F2 fragments are linked together by two disulfide bonds. Uncut F0 protein without a signal peptide sequence or F1-F2 heterodimers can form RSV F protomers. Three such protomers assemble to form the final RSV F protein trimer.

[0008] The F protein of subtypes A and B shares approximately 90% amino acid sequence similarity and is the main cross-protective antigen. Therefore, neutralizing antibodies induced by the F protein can simultaneously inhibit viral infection from both subtypes A and B. An example sequence of the F0 precursor polypeptide of subtype A is provided in SEQ ID NO:1 (A2 strain; Swiss Prot / uniprot P03420), and another example sequence of the F0 precursor polypeptide of subtype A is provided in SEQ ID NO:4 (ON1 strain; GenBank: QYW11910; Swiss Prot / uniprot A0A0B4L8X2). An example sequence of the F0 precursor polypeptide of subtype B is provided in SEQ ID NO:7 (BA9 strain; Genebank: VVF90619.1, uniprot: A0A482JPF1).

[0009] The F protein on the surface of RSV is a major target for neutralizing antibodies. The F protein promotes viral fusion with the host cell membrane, forming a typical syncytia. The RSV F protein has a trimer structure and mediates the fusion of the viral envelope and host cell through dramatic conformational changes. Before fusion initiation, the F protein is in a pre-fusion conformation (preF), which is unstable and has a low energy barrier. When very close to the host cell, the fusion peptide (FP) inserts into the host cell membrane, allowing the F protein to cross both the viral envelope and the host cell membrane. Subsequently, the F protein forms a hairpin structure in the trimer, connecting the two membranes and promoting fusion, forming a highly stable post-fusion conformation (postF). The pre-fusion F protein trimer is lollipop-shaped, while the post-fusion F protein is cane-shaped; the two conformations are structurally very different and have different antigenic epitopes. Ideally, to prevent viral entry, drugs should be developed using the antigenic epitopes of the preF conformation. However, the preF conformation of the F protein is a metastable structure located at a higher energy level. Over time, it transforms into a low-energy, nonfunctional postF form. Furthermore, exposure to hydrophobic fusion peptides makes it prone to aggregation, and exposure to high temperatures or low pH conditions can lead to the preF conformation transforming into the postF conformation (McLellan, JS, et al. 2013). Many subunit vaccines or vector vaccines based on postF as an immunogen have been discontinued due to low neutralizing antibody activity and insufficient protective efficacy. Therefore, the postF conformation should be avoided as a vaccine immunogen. RSV preF protein induces higher levels of neutralizing antibodies and more activated memory B cells than postF protein, making it the optimal choice for vaccine antigen.

[0010] Through antibody competition and structural studies of the F protein-antibody complex, the surface of the F protein was divided into six non-overlapping domains, leading to the identification of six neutralizing antibody epitopes on the F protein: I, II, III, IV, V, and... Epitopes, where V and The epitopes are specific to the pre-conformation F protein. Epitopes I-IV coexist in both the pre- and post-conformation F proteins. Antibodies targeting epitopes II and IV bind similarly to both pre- and post-conformation F proteins, while antibodies targeting epitopes III and I preferentially bind to the pre- and post-conformation F proteins, respectively (Graham, BS 2017). Studies have shown that epitopes V and IV specifically target preF. Epitopes are high-level epitopes and target epitopes. Neutralizing antibodies targeting epitopes, such as D25, AM22, and 5C4, exhibit 10–100 times greater neutralizing activity than Palivizumab, an antibody targeting epitopes II (McLellan, JS, et al. 2013) (Ngwuta, JO, et al. 2015). Human serum primarily contains neutralizing antibodies against preF, and... Epitope antibody levels are positively correlated with neutralization levels (Ngwuta, JO, et al. 2015). To address the instability of preF, several marketed RSV vaccines using F as the antigen employ different strategies to maintain the F protein in a stable pre conformation (Che, Y., et al. 2023; CN105473604A; CN115103682A). The stable pre conformation of the F protein can induce high levels of antibodies against V and... Epitope-neutralizing antibodies are currently a focus of RSV vaccine development. Therefore, it is necessary to build upon previous experience and further improve the stability and immunogenicity of the preF protein through structure-based rational design. Summary of the Invention

[0011] The purpose of this disclosure is to provide an RSV F protein with improved stability and immunogenicity.

[0012] The first aspect of this disclosure provides a recombinant respiratory syncytial virus (RSV) F protein mutant comprising an F2 polypeptide and an F1 polypeptide from the N-terminus to the C-terminus, wherein the mutant comprises the following mutations relative to the wild-type RSV F protein sequence: 55V (preferred) / A, 227T (preferred) / L / I / M, and 405V, wherein the RSV F position corresponds to the amino acid sequence of the reference RSV F0 polypeptide shown in SEQ ID NO:1.

[0013] In some embodiments, the mutants disclosed herein also contain 185E or 190V (preferred).

[0014] In some implementations, the mutants disclosed herein also contain 290G.

[0015] In some embodiments, the mutants of this disclosure contain the following mutations relative to the wild-type RSV F protein sequence: S55V (preferred) / A, N227T (preferred) / L / I / M, and S405V.

[0016] In some embodiments, the mutants disclosed herein also include V185E or S190V (preferred).

[0017] In some implementations, the mutants disclosed herein also include S290G.

[0018] In some embodiments, the mutants disclosed herein do not contain the pep27 peptide.

[0019] In some embodiments, the mutants disclosed herein do not contain furin restriction sites.

[0020] In some embodiments, the F1 peptide in the mutant of this disclosure does not contain transmembrane and intracellular domains.

[0021] In some embodiments, the mutants disclosed herein do not contain the pep27 peptide, and the F2 peptide is directly linked to the F1 peptide.

[0022] In some embodiments, the mutant of this disclosure does not contain the pep27 peptide, and the F2 peptide and the F1 peptide are linked by a heteropeptide linker. Preferably, the linker is selected from: SGS (preferred), SSG, SPS, SGSG (SEQ ID NO:42), SGGS (SEQ ID NO:41), SGGG (SEQ ID NO:45), SGG, S, GSP, GSGS (SEQ ID NO:43), GSG, GSAS (SEQ ID NO:40), GS, GGSGG (SEQ ID NO:44), GGSG (SEQ ID NO:46), GGS, GGGS (SEQ ID NO:39) or G.

[0023] In some embodiments, the F2 and F1 peptides in the mutant of this disclosure contain positions 26-104 or 26-108 and positions 145-513 or 138-513 of the RSV F protein, respectively.

[0024] Preferably, the F2 polypeptide of this disclosure has the sequence shown in SEQ ID NOs:11, 14, 17, 20, 23, 26, 29 or 32, or the F2 polypeptide sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the sequence shown in SEQ ID NOs:11, 14, 17, 20, 23, 26, 29 or 32, provided that the F2 polypeptide contains the following mutation: S55V / A.

[0025] Preferably, the F1 polypeptide of this disclosure has the sequence shown in SEQ ID NOs:12, 15, 18, 21, 24, 27, 30 or 33, or the F1 polypeptide sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the sequence shown in SEQ ID NOs:12, 15, 18, 21, 24, 27, 30 or 33, provided that the F1 polypeptide contains the following mutations: N227T / L / I / M and S405V, preferably also containing V185E or S190V, more preferably also containing S290G.

[0026] In some embodiments, the F2 and F1 peptides in the mutant of this disclosure comprise positions 26-104 and 145-513 of the RSV F protein, respectively.

[0027] Preferably, the F2 polypeptide of this disclosure has the sequence shown in SEQ ID NO:11 or 23, or the F2 polypeptide has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:11 or 23, provided that the F2 polypeptide contains the following mutation: S55V.

[0028] Preferably, the F1 polypeptide of this disclosure has the sequence shown in SEQ ID NO:12 or 24, or the F1 polypeptide has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:12 or 24, provided that the F1 polypeptide contains the following mutations: N227T and S405V, preferably also containing S190V, and more preferably also containing S290G.

[0029] In some embodiments, the mutants of this disclosure further comprise a signal peptide, preferably a sequence selected from the sequences shown in SEQ ID NOs:34-36.

[0030] In some embodiments, the C-terminus of the F1 polypeptide of the present disclosure mutant is linked to a polymerizing domain (preferably a trimerizing domain).

[0031] Preferably, the polymerized domain connected to the C-terminus comprises a T4-foldon trimerized domain; and / or

[0032] Preferably, the T4-foldon trimerization domain comprises the amino acid sequence shown in SEQ ID NO:37; and / or

[0033] Preferably, the polymerizing domain (such as the T4-foldon trimerizing domain) is linked to amino acid residue 513 of the RSV F protein mutant; and / or

[0034] Preferably, the multimerizing domain (such as the T4-foldon trimerizing domain) is directly or through a peptide linker (such as the SAIG ​​(SEQ ID NO:38) tetrapeptide) linked to the F1 polypeptide.

[0035] In some embodiments, the mutants of this disclosure exhibit increased stability under heat treatment at 60°C for 20 min compared to the corresponding wild-type RSV F protein, wherein said stability is associated with specific recognition. The binding of epitopes to antibodies (such as combined antibodies against D25, AM22, and / or RSD5) is measured.

[0036] In some embodiments, the mutants of this disclosure exhibit increased stability at pH 2.5 for 20 min compared to the corresponding wild-type RSV F protein, wherein said stability is measured by the binding of the mutant to a preF-specific antibody (such as a combination antibody of D25, AM22 and / or RSD5).

[0037] In some embodiments, the mutant of this disclosure induces a comparable or higher humoral and / or cellular immune response compared to wild-type RSV F protein, when administered to a subject at the same dose of RSV F antigenic peptide. Preferably, the induced humoral immune response is increased by at least 3-6 times.

[0038] In some embodiments, the mutants of this disclosure comprise the sequence shown in SEQ ID NO:10 or 22, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:10 or SEQ ID NO:22, provided that the mutant comprises the following mutations: S55V, N227T, and S405V, preferably also comprising S190V, and more preferably also comprising S290G.

[0039] In some implementations, the wild-type RSV is subtype A or subtype B.

[0040] In some implementations, the mutants of this disclosure are in the form of trimers.

[0041] The second aspect of this disclosure provides a nucleic acid molecule encoding a mutant of this disclosure.

[0042] In some implementations, the nucleic acid molecule is mRNA (preferably) or DNA.

[0043] A third aspect of this disclosure provides a vector comprising the nucleic acid molecules described in the second aspect of this disclosure.

[0044] The fourth aspect of this disclosure provides a host cell comprising the nucleic acid molecule described in the second aspect of this disclosure or the vector described in the third aspect.

[0045] A fifth aspect of this disclosure provides an immunogenic composition comprising the recombinant RSV F protein mutant of the first aspect of this disclosure, the nucleic acid molecule of the second aspect, or the vector of the third aspect.

[0046] In some embodiments, the immunogenic compositions of this disclosure further comprise an adjuvant.

[0047] In some embodiments, the adjuvants used in this disclosure are selected from Th1 adjuvants or aluminum adjuvants;

[0048] Preferably, the Th1 adjuvant is selected from:

[0049] (1) Oil-in-water emulsion adjuvants, such as (a) MF59; (b) SAF; or (c) Ribi™ adjuvant system (RAS);

[0050] (2) Saponin adjuvants, such as Quil A or QS-21;

[0051] (3) Bacterial lipopolysaccharides, such as AGP;

[0052] (4) Synthesize polynucleotides, such as oligonucleotides containing CpG motifs;

[0053] (5) Cytokines, such as interleukins, interferons, GM-CSF, M-CSF, TNF, or co-stimulatory molecules B7-1 and B7-2,

[0054] The interleukin is preferably IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, or IL-18, and the interferon is preferably gamma interferon;

[0055] (6) Mucosal adjuvants; and

[0056] (7) Other substances that act as immunostimulants to enhance the effectiveness of the composition.

[0057] Preferably, the aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.

[0058] In some embodiments, the immunogenic compositions disclosed herein are liquids or lyophilized products.

[0059] A sixth aspect of this disclosure provides a vaccine comprising the recombinant RSV F protein mutant described in the first aspect of this disclosure, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, or the immunogenic composition described in the fifth aspect.

[0060] The seventh aspect of this disclosure provides a method for treating or preventing diseases or symptoms caused by RSV using the recombinant RSV F protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the immunogenic composition described in the fifth aspect, or the vaccine described in the sixth aspect of this disclosure.

[0061] The eighth aspect of this disclosure provides the recombinant RSV F protein mutant described in the first aspect of this disclosure, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the immunogenic composition described in the fifth aspect, or the vaccine described in the sixth aspect of this disclosure for the treatment or prevention of diseases or symptoms caused by RSV. Attached Figure Description

[0062] Figure 1 illustrates the contribution of the preferred molecular protein mutation sites of strain A2 to the stability of preF. In the figure: black background indicates sites of reversion mutations, i.e., retaining the same amino acid type as the wild type; Pfizer RSV-F (PF-847) design reference: Doi:10.1126 / scitranslmed.ade6422 mutant 847; GSK RSV-F design reference: patent CN105473604A.

[0063] Figure 2 illustrates the contribution of the preferred molecular protein mutation sites of BA9 strain to preF stability. In the figure: black background indicates sites of reversion mutations, i.e., retaining the same amino acid type as the wild type; Pfizer RSV-F (PF-847) design reference: Doi:10.1126 / scitranslmed.ade6422 mutant 847; GSK RSV-F design reference: patent CN105473604A.

[0064] Figure 3 shows the SEC-HPLC chromatograms of the RSF F protein mutants A2F protein (Figure 3A) and BA9F protein (Figure 3B).

[0065] Figure 4 shows the binding of neutralizing antibodies to EC at different epitopes of A2F and BA9F proteins. 50Among them, A: the binding activity of A2F and BA9F trimer proteins to antibody D25; B: the binding activity of A2F and BA9F trimer proteins to antibody D25; C: the binding activity of A2F and BA9F trimer proteins to antibody D25.

[0066] Figure 5 shows the ratio of preF and postF antibody titers induced by RSV preF and postF protein mouse immune serum.

[0067] Figure 6 shows the neutralizing titers of RSV A2 virus strain in the serum of mice immunized with RSV preF and postF proteins. Detailed Implementation

[0068] definition

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Generally, the terms used herein are those well-known and commonly used in the art.

[0070] The terms “a” and “an” mean one or more, and the term “or” means and / or, where the context allows. Therefore, as used in the specification and appended claims, the singular forms “a,” “an,” and “the” include the plural objects unless the context clearly indicates otherwise.

[0071] "Amino acid difference," "residue difference," or "amino acid mutation" refers to a change in residues at a specific position in a polypeptide sequence when compared to a reference sequence. The position of a polypeptide sequence where a specific amino acid or amino acid change ("residue difference") exists is sometimes described herein as "X". n " or "position n", where n refers to the residue position relative to the reference sequence, and "X" represents an amino acid residue.

[0072] As disclosed herein, a polypeptide sequence may contain one or more residue differences relative to a reference sequence, wherein multiple residue differences are typically indicated by specific positions where changes are made relative to the reference sequence (e.g., “one or more residue differences at the following residue positions compared to SEQ ID NO:1: S55, V185, S190, N227, S290, or S405”).

[0073] Specific substitution mutations, where a specific residue in a reference sequence is replaced by a different designated residue, can be represented by the conventional symbol "X(number)X'", where X is a single-letter identifier of the residue in the reference sequence, "number" is the position of the residue in the reference sequence, and X' is a single-letter identifier of the residue substitution in the engineered sequence. An example is the S55V mutation.

[0074] The term "ectodomain" refers to the portion of a full-length polypeptide that extends beyond the viral envelope. Polypeptides are known to typically contain an intracellular domain, a transmembrane domain, and the remainder an extracellular domain ("ECD"). When used herein, the term "ectodomain" or "ECD" refers to the amino acids of a polypeptide in wild-type form that extend beyond the viral envelope, or any portion thereof that can be recognized by antibodies. Therefore, an extracellular domain includes the entire viral extra-enveloped domain, or any number of residues suitable for recombinant expression and included in an antigen composition, including 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the entire wild-type extracellular domain of the polypeptide. That is, the viral extra-enveloped domain can be shortened or truncated by methods known in the art to remove foreign domains on the carboxyl-terminus or amino-terminus, or both, of the polypeptide as needed, to obtain more efficient and robust expression of the viral extra-enveloped domain of the polypeptide.

[0075] The term "full length" for a given polypeptide refers to the polypeptide form naturally translated from the DNA sequence encoding the first methionine in the amino acid sequence, starting with the ATG start codon and ending with the TGA, TAG, or TTA stop codon or any stop codon used by the organism.

[0076] "Conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally involves replacing amino acids in a polypeptide with amino acids from the same or similarly defined amino acid classes. However, as used herein, in some embodiments, a conserved mutation does not include substitutions from hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small to small residues, if the conserved mutation may alternatively be a substitution from aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restricted to restricted residues. Furthermore, as used herein, A, V, L, or I can be conservedly mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions in this paper include: (1) for residues A, L, V, I, possible conservative mutations are: (a) other aliphatic residues (A, L, V, I), (b) other nonpolar residues (A, L, V, I, G, M); (2) for residues G, M, possible conservative mutations are: other nonpolar residues (A, L, V, I, G, M); (3) for residues D, E, possible conservative mutations are: other acidic residues (D, E); (4) for residues K, R, possible conservative mutations are: other basic residues (K, R); (5) for residues N, Q, S, T, possible conservative mutations are: other polar residues; (6) for residues H, Y, W, F, possible conservative mutations are: other aromatic residues (H, Y, W, F).

[0077] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "referring to," or "relative to" means the residue number of the reference sequence specified when comparing a given amino acid or polynucleotide sequence with a reference sequence.

[0078] Given the conservation of RSV F sequences, those skilled in the art can readily compare the amino acid positions between different native RSV F sequences to identify the corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, the furin cleavage site falls at the same amino acid position in almost all identified native RSV F0 precursor proteins. Therefore, the conservation of RSV F protein sequences between different strains and subtypes allows the use of a reference RSV F sequence to compare amino acids at specific positions in RSV F proteins. For the purposes of this disclosure (unless the context otherwise requires), the reference F0 protein precursor polypeptide shown in SEQ ID NO:1 gives the RSV F protein amino acid positions.

[0079] In this disclosure, the "reference sequence" is not limited to the wild-type sequence, but may also include engineered or modified sequences. For example, in some embodiments, the "reference sequence" may be a previously engineered or modified amino acid sequence.

[0080] The methods for comparing a sequence with a prescribed reference sequence are known to those skilled in the art. For example, the Needleman-Wunsch method can be used to compare any amino acid or polynucleotide sequence with a reference sequence.

[0081] "Corresponding amino acid position" is a term widely used and familiar to technical personnel. Any well-known amino acid alignment method can be used to align amino acid sequences to determine the corresponding amino acid position. For example, the NCBI BLAST algorithm can be used to determine the corresponding amino acid position.

[0082] Several subtypes of RSV exist, including human subtype A, human subtype B, and bovine subtype. Within each RSV subtype, there are individual strains of each subtype.

[0083] The RSV fusion (F) protein described in this article refers to the RSV envelope glycoprotein that promotes the fusion of the virus and the cell membrane. In nature, the RSV F protein is initially synthesized as a single polypeptide precursor of approximately 574 amino acids, called F0. F0 includes an N-terminal signal peptide that directs to the endoplasmic reticulum, wherein the signal peptide (approximately the first 25 residues of F0) is proteased and cleaved. The remaining F0 residues oligomerize to form a trimer, which is further proteased by cellular proteases at two conserved furin cleavage sequences (approximately F0 positions 109 and 136) to produce two disulfide-linked fragments, F1 and F2. F2 originates from the N-terminal portion of the F0 precursor and comprises approximately residues 26-109 of F0. F1 comprises the C-terminal portion of the F0 precursor (approximately residues 137-574), which includes an extracellular / luminal region (approximately residues 137-524), a transmembrane domain (approximately residues 525-550), and a cytoplasmic domain at the C-terminus (approximately residues 551-574).

[0084] RSV F1 polypeptide (F1): The peptide chain of the RSV F protein. As used herein, “F1 polypeptide” refers to the native F1 polypeptide and modified F1 polypeptides that include modifications to the native sequence (e.g., amino acid substitutions, insertions, or deletions), such as those engineered to stabilize recombinant F proteins (including the modified F1 polypeptide) in the pre-fusion conformation of the RSV F protein. The native F1 comprises approximately residues 137-574 of the RSV F0 precursor and includes (from N-terminus to C-terminus) an extracellular / luminal region (approximately residues 137-524), a transmembrane domain (approximately residues 525-550), and a cytoplasmic domain (approximately residues 551-574). Several embodiments include F1 polypeptides modified compared to the native F1 sequence, such as lacking the transmembrane and cytoplasmic domains and / or including one or more amino acid substitutions that stabilize the recombinant F protein (containing the F1 polypeptide) in the pre-fusion conformation. In one example, the disclosed RSV F protein includes an F1 polypeptide with the loss of a transmembrane domain and a cytoplasmic domain and substitutions of several amino acid residues. In several embodiments, the F1 polypeptide includes a C-terminus linked to a trimerized domain.

[0085] RSV F2 polypeptide (F2): The polypeptide chain of the RSV F protein. As used herein, “F2 polypeptide” refers to the native F2 polypeptide and modified F2 polypeptides that include modifications to the native sequence (e.g., amino acid substitutions), such as those designed to stabilize recombinant F proteins (including the modified F2 polypeptide) in the pre-fusion conformation of the RSV F protein. The native F2 comprises approximately residues 26-109 of the RSV F0 precursor. In the native RSV F protein, the F2 polypeptide is linked to the F1 polypeptide via two disulfide bonds.

[0086] RSV pep27 polypeptide (pep27): A 27-amino acid polypeptide cleaved from the F0 precursor during the maturation of the RSV F protein. pep27 has two furin cleavage sites on its side, and it is cleaved by cellular proteases during F protein maturation to produce F1 and F2 polypeptides.

[0087] Pre-fusion conformation of RSV F protein: The structural conformation adopted by RSV F protein prior to triggering a fusion-promoting event that leads to RSV F transitioning to its post-fusion conformation and becoming the mature RSV F protein in the secretory system after processing. In the pre-fusion state, RSV F protein includes an antigenic site at the distal apex of the membrane (“antigen site”). The recombinant RSV F protein, which includes RSV F residues 62-69 and 196-209, and also includes epitopes of antibodies such as D25, AM22, and RSD5, can be specifically bound to antibodies that are specific to the pre-fusion conformation of RSV F protein (e.g., antibodies that specifically bind to epitopes within the antigenic site, such as D25, AM22, or RSD5 antibodies) as used herein. Other pre-fusion specific antibodies include, but are not limited to, 5C4 and MPE8 antibodies.

[0088] Post-fusion conformation of RSV F protein: The structural conformation adopted by the RSV F protein, which is not in its pre-fusion conformation, wherein the N-terminus and C-terminus of the RSV F protein are close together in a stable helix-helix configuration. The post-fusion conformation of RSV F protein has been described at the atomic level (see, for example, McLellan, JS, et al. 2013; Swanson, KA, et al. 2011; and structural coordinates stored with PDB accession number 3RRR). The post-fusion conformation of RSV F protein is similar to that of other known paramyxoviral glycoproteins, including PIV5 F protein. In the post-fusion conformation, the RSV F protein does not include the antigenic site. And therefore it is not specifically bound by D25, AM22 or RSD5.

[0089] The three F2-F1 promeromers oligomerize in the mature F protein in a metastable "pre-fusion" conformation, which is triggered to undergo a conformational change (becoming the "post-fusion" conformation) upon contact with the target cell membrane. This conformational change exposes a hydrophobic sequence called the fusion peptide, located at the N-terminus of the F1 polypeptide, which associates with the host cell membrane and promotes the fusion of the viral or infected cell membrane with the target cell membrane.

[0090] Single-chain RSV F protein: Recombinant RSV F protein expressed as a single polypeptide chain comprising RSV F1 and RSV F2 polypeptides. The single-chain RSV F protein trimerizes to form the extracellular domain of the RSV F protein. The single-chain RSV F protein does not include the furin cleavage site of the pep27 polypeptide flanking the RSV F protein; therefore, when produced in the cell, the F0 polypeptide does not cleave into separate F1 and F2 polypeptides. In several embodiments, the F1 and F2 polypeptides are linked by a linker such as a peptide linker.

[0091] Unfavorable electrostatic interactions in the pre-fusion or pre-fusion trimer configuration can be identified by methods known in the art, such as visually examining the crystal structure of RSV F in the pre-fusion or pre-fusion trimer configuration, or by using computational protein design software (such as BioLuminate). TM [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio TM [Discovery Studio Modeling Environment,Accelrys,San Diego,2015], MOE TM [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015] and Rosetta TM [Rosetta, University of Washington, Seattle, 2015]).

[0092] As used herein, the word "comprising" is open-ended and means "including but not limited to". "Having" is used herein as a synonym for "comprising". It should be understood that the use of the word "comprising" to describe embodiments herein encompasses those embodiments described as "consisting of" and / or "substantially consisting of".

[0093] As used herein, “immunogenicity” refers to the ability of an antigen to induce an immune response. As used herein, “improved immunogenicity” means that, when administered to a subject, the modified RSV F protein elicits a stronger RSV-specific immune response than the unmodified RSV F protein. In some embodiments, the modified RSV F protein elicits a comparable or stronger T-cell or B-cell response compared to the unmodified RSV F protein.

[0094] "Heterologous" polynucleotides refer to any polynucleotide introduced into host cells through laboratory techniques, including polynucleotides that are removed from host cells, manipulated in the laboratory, and then reintroduced into host cells.

[0095] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other naturally occurring contaminants (e.g., proteins, lipids, and polynucleotides). The term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis). Improved antigenic peptides can be present intracellularly, in cellular media, or prepared in various forms, such as lysates or isolated formulations. Therefore, in some embodiments, the improved antigenic peptide may be an isolated polypeptide.

[0096] "Sequence identity percentage," "percentage identity," and "identity percentage" are used in this document to refer to comparisons between polynucleotide or polypeptide sequences and are determined by comparing two best-aligned sequences within a comparison window. The polynucleotide or polypeptide sequence portion within the comparison window may contain additions or deletions (i.e., vacancies) to achieve optimal alignment of the two sequences compared to a reference sequence. The percentage is calculated by determining the number of identical nucleic acid bases or amino acid residues appearing in both sequences, or the number of positions where nucleic acid bases or amino acid residues align with vacancies, to produce the number of matching positions. The number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to produce the percentage of sequence identity. Best-alignment and percentage sequence identity determinations are performed using the BLAST and BLAST 2.0 algorithms. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information website.

[0097] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to polymers of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, esterification, myristilation, ubiquitination, etc.). This definition includes D- and L-amino acids, as well as mixtures of D- and L-amino acids.

[0098] As used herein, “nucleic acid” refers to a polymer of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or analogues thereof. It includes DNA, RNA, and DNA / RNA hybrids. It also includes DNA or RNA analogues, such as those containing modified backbones (e.g., peptide nucleic acids (PNAs) or modified bases). Therefore, nucleic acids in this disclosure include mRNA, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, etc. “Nucleic acid sequence” as used herein may also include, but is not limited to, native or foreign sequences, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators.

[0099] Nucleic acids can be in recombinant form, that is, in a form that does not exist in nature. For example, a nucleic acid can contain one or more heterologous nucleic acid sequences (e.g., sequences encoding another antigen and / or control sequences such as promoters or internal ribosome entry sites). Nucleic acids can be part of a vector, that is, part of a nucleic acid designed to transduce / transfect one or more cell types. A vector can be, for example, an "expression vector" designed to express a nucleotide sequence in a host cell, or a "viral vector" designed to produce recombinant viruses or virus-like particles.

[0100] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence that enables the expression of the DNA sequence in a suitable host. A vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into its genome. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. For the purposes of this disclosure, plasmid vectors are preferred. Typical plasmid vectors used for this purpose have the following structures: (a) an origin of replication that allows efficient replication, enabling the generation of hundreds of plasmid vectors per host cell; (b) an antibiotic resistance gene that allows selection of host cells transformed with the plasmid vector; and (c) a restriction enzyme site that allows the insertion of a foreign DNA fragment. Even if a suitable restriction enzyme site is not present in the vector, the vector can be easily ligated to foreign DNA using synthetic oligonucleotide adaptors or linkers according to conventional methods.

[0101] As used herein, the term "recombinant vector" generally refers to a recombinant vector in which a foreign DNA fragment, typically in the form of a double-stranded DNA fragment, is inserted. Here, foreign DNA refers to exogenous DNA that is not naturally found in the host cell. Once the recombinant vector enters the host cell, it can replicate independently of the host chromosomal DNA, thereby producing several copies of the vector and the inserted (foreign) DNA.

[0102] After ligation, the gene or recombinant vector is transformed or transfected into host cells. For “transformation” or “transfection”, several types of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, examples of which include electroporation, calcium phosphate precipitation, DEAE-glucan transfection, and lipid transfection.

[0103] As is well known in the art, in order to increase the expression level of the transfected gene in the host cell, the gene in question should be operatively linked to the transcriptional and translational expression control sequences in the selected expression host to perform its function.

[0104] As used herein, the term "transformation" refers to the introduction of DNA into a host so that the DNA can replicate as an extrachromosomal factor or through chromosomal integration. It should be understood, of course, that not all vectors have the same function when expressing the gene sequences of this disclosure. Similarly, not all host cells have equivalent function relative to the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences, and hosts without departing from the spirit of this disclosure and without excessive experimental burden. For example, the host must be considered when selecting a vector, as it must replicate within it. In this regard, the copy number of the vector, its ability to regulate copy number, and the expression of other proteins encoded by the vector must also be considered.

[0105] In this disclosure, the host cells to be transformed include prokaryotic or eukaryotic cells; preferably selected from, but not limited to, the following groups: animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0106] The host cells can be derived from plants or mammals. Preferred examples may include monkey kidney cells (COS-7 cells), NS0 cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK293 cells, with CHO cells being preferred.

[0107] As used in this article, the term "prevention" refers to suppressing the occurrence of a condition or disease in subjects who have not yet been diagnosed with it and are likely to develop it.

[0108] As used herein, the term “treatment” means (a) inhibiting the progression of a condition or disease or its symptoms; (b) alleviating a condition or disease or its symptoms; or (c) eliminating a condition or disease or its symptoms. In this specification, the terms “treatment” or “therapeutic agent” also include the meaning of “adjunctive therapy” or “therapeutic aid”.

[0109] As used herein, the term "active ingredient" refers to a vaccine composition sufficient to produce the desired effect.

[0110] As used herein, the term “immune response” refers to cell-mediated (T cell) immune responses and / or antibody (B cell) responses.

[0111] In this disclosure, the vaccine composition is characterized in that it further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0112] The vaccine compositions of this disclosure can be prepared in unit dosage forms or in the form of multi-dose containers by means of methods readily implemented by those skilled in the art to which this disclosure pertains, using pharmaceutically acceptable carriers and / or excipients.

[0113] In this disclosure, the vaccine composition is characterized by further comprising an adjuvant. Typically, a protein antigen alone cannot strongly induce an immune response; therefore, mixing it with an adjuvant can enhance the efficacy of the vaccine composition.

[0114] As used herein, the term "adjuvant" refers to a substance, including reagents, molecules, etc., that nonspecifically promotes an immune response to an antigen during the initial activation of immune cells. Similarly, each is not an immunogen of the host but enhances immunity by increasing the activity of cells in the immune system. The adjuvants used in this disclosure that can enhance the immune response can be administered simultaneously with the vaccine composition or sequentially at time intervals.

[0115] The adjuvants disclosed herein are characterized in that they are selected from, but not limited to, Th1 adjuvants and / or aluminum adjuvants and combinations thereof, wherein the Th1 adjuvants may be:

[0116] Oil-in-water emulsion adjuvants, such as (a) MF59; (b) SAF; or (c) Ribi™ adjuvant system (RAS);

[0117] Saponin adjuvants, such as Quil A or QS-21;

[0118] Bacterial lipopolysaccharides, such as AGP;

[0119] Synthesize polynucleotides, such as oligonucleotides containing CpG motifs;

[0120] Cytokines, such as interleukins, interferons, GM-CSF, M-CSF, TNF, or co-stimulatory molecules B7-1 and B7-2,

[0121] The interleukin is preferably IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, or IL-18, and the interferon is preferably gamma interferon;

[0122] Mucosal adjuvants; and

[0123] Other substances that serve as immunostimulants to enhance the effectiveness of the composition;

[0124] The aluminum adjuvant is aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.

[0125] The optimal dosage of the vaccine composition disclosed herein can be determined through standard studies, which include observing an appropriate immune response in subjects. Following the initial vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

[0126] The appropriate dosage of the vaccine composition disclosed herein depends on factors such as the method of formulation, route of administration, patient's age, weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and responsiveness, and can be appropriately determined by those skilled in the art by taking these factors into consideration.

[0127] The vaccine composition disclosed herein can be administered via methods commonly used in the medical field.

[0128] The present disclosure will be described in more detail below by way of examples. These examples are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the present disclosure is not to be construed as limited to these examples.

[0129] Example

[0130] Example 1: Design and preparation of RSV F protein mutants

[0131] In the following examples, the amino acid mutation positions are all relative to the coding sequence in the natural F0 protein. Unless otherwise specified, the protein construction uses the C-terminus (amino acid 513) of the extracellular region of the natural protein to introduce T4-foldon. The C-terminus and T4-foldon are linked by the tetrapeptide linker SAIG ​​(SEQ ID NO:38). The pep27 peptide between F2 and F1 is removed, and part of the fusion peptide (FP) at the N-terminus of the F1 peptide is removed. The F2 and F1 peptides are linked by different linkers. In the following text, SC, SC11, SC12, etc., indicate different types of linkers, and M1, M2, and M83, etc., indicate artificially designed mutants.

[0132] Construction of RSV F mutant expression vector

[0133] Wild-type and mutant initial templates were obtained through whole-genome synthesis. The target gene was then obtained through multiple amplification and mutagenesis using polymerase chain reaction (PCR) with TKs Gflex DNA Polymerase (Takara) via primer design. The resulting product was then inserted into a mammalian expression vector using the In-Fusion HD Cloning Kit (Takara). Successful construction of the expression vector was verified by DNA sequencing. The constructed expression plasmid was transfected into 293E or CHO cells for target gene expression.

[0134] For all commercial kits or reagents, follow the manufacturer's instructions.

[0135] Expression and purification of RSV F mutant

[0136] Cells and culture supernatant were separated by centrifugation, and the culture supernatant was then loaded onto a cation exchange column. The target product was eluted with increasing salt concentrations. The eluted fraction was incubated with a surfactant to inactivate the virus, and after inactivation, it was loaded onto an anion exchange chromatography column, where the target product was eluted with increasing salt concentrations. The eluted target product was then ultrafiltered to a final storage buffer. The supernatant and purified protein were used in the in vitro and in vivo assays described herein.

[0137] RSV preF protein immunogenicity stability screening method

[0138] RSV preF protein expression supernatant optimization screening was performed using a set of monoclonal antibodies to assess structural integrity and pre-fusion conformation stability (see Table 1), including antibodies that specifically bind to the preF protein. Epitope monoclonal antibody mixture (D25, AM22, and RSD5) was used for characterization Epitope conformation integrity; specific binding preF protein trimer monoclonal antibody (AM14) to characterize trimer formation; and specific binding postF protein monoclonal antibody (4D7) to characterize postF protein expression level.

[0139] RSV preF protein mutants were transiently transfected into CHO or 293E cells. The expressed target protein was secreted into the culture supernatant. The culture supernatant containing preF protein was subjected to heat shock at 60°C or acid treatment (pH 2.5). The supernatant culture medium before and after treatment was used to detect the binding of different conformation-specific antibodies by ELISA to evaluate the conformational stability of preF. By comparing the binding ability between different mutants and the percentage of binding retention after heat shock treatment, mutant molecules with higher stability were screened.

[0140] The stability of the designed RSV F protein mutant was evaluated using a heat shock test. During the heat shock test, the supernatant of the designed mutant was incubated at 60°C for 20 min and analyzed using pre-fusion assays in an ELISA test. Detection of epitope-specific monoclonal antibodies D25, AM22, RSD5, and pre-fusion trimer-specific antibody AM14. The ratio of antibody reactivity of heat-shocked samples to non-heat-shocked samples is defined as the heat shock resistance parameter. More stable mutants are expected to have higher heat shock resistance. Sample heat shock resistance % = OD 处理样品 / OD 未处理样品 ×100%, of which OD 处 理样品This indicates the OD values ​​of the samples after heat shock treatment at 60℃ for 20 minutes, showing the binding of the samples to antibodies at different epitopes. 未处理样品 This indicates the OD value of an untreated sample bound to antibodies at different epitopes.

[0141] The stability of the designed RSV F protein mutant was evaluated by a low-pH incubation experiment at room temperature. During the low-pH tolerance test, the supernatant of the designed mutant was incubated at a low pH of 2.5 for 20 min and analyzed by ELISA using the pre-fusion assay. Epitope-specific monoclonal antibodies D25, AM22, RSD5, and pre-fusion trimer-specific antibody AM14 were detected. During pH tolerance assays, the reactivity of the pre-fusion antibody in the supernatant after incubation at pH 2.5 for 20 min was compared with that in the fresh culture supernatant. The activity ratio was defined as the low pH tolerance of the mutant. More stable mutants were expected to have higher tolerance to low pH. Low pH tolerance (%) = OD 处理样品 / OD 未处理样品 ×100%, of which OD 处理样品 This indicates the OD values ​​of samples after low pH 2.5 incubation for 20 minutes and binding to different epitope antibodies. 未处理 样品 This indicates the OD value of an untreated sample bound to antibodies at different epitopes.

[0142] The OD value of the PostF conformation (4D7 antibody binding to I epitope) can characterize the expression level of postF protein. Fresh supernatant is detected by I epitope-specific monoclonal antibody in ELISA assay. The measured OD value represents the OD value of the untreated sample bound to different epitope antibodies. The higher the OD value, the higher the content of postF.

[0143] Table 1. Specific monoclonal antibodies used to evaluate the conformational stability of RSV-F trimer.

[0144] 1.1 RSV preF protein mutation design

[0145] The F protein sequence of subtype A was selected based on amino acids 1-513 of the extracellular region of the F0 protein (uniprot: P03420) (SEQ ID NO: 1) of strain A2. The C-terminus and T4-foldon were linked by the tetrapeptide linker SAIG ​​(SEQ ID NO: 38). The pep27 peptide and part of the fusion peptide were removed from F1 and F2, and they were linked by the linker SC linker (positions 109-137 of the native F0 protein were replaced by SC linker: GGSGSGRS (SEQ ID NO: 47)), named A2-RSV-F(SC) (SEQ ID NO: 49).

[0146] To enhance the neutralizing effect against the current subtype A strain, after sequence alignment with the F0 protein of the ON1 strain (genbank: QYW11910) (SEQ ID NO:4), five sites on A2-RSV-F(SC) were mutated to the corresponding amino acid types (P102A, T103A, N276S, I379V, M447V) of the ON1 strain, and named A2-RSV-F(SC-M1) (SEQ ID NO:50).

[0147] The F protein sequence of the BA9 subtype was selected based on amino acids 1-513 of the extracellular region of the BA9 strain F0 protein (Genebank: VVF90619.1) (SEQ ID NO:7). The C-terminus and T4-foldon were linked by the tetrapeptide linker SAIG ​​(SEQ ID NO:38). The pep27 peptide and part of the fusion peptide were removed from F1 and F2, and they were linked by the linker SC linker (positions 109-137 of the native F0 protein were replaced by the SC linker: GGSGSGRS (SEQ ID NO:47)), named BA9-RSV-F(SC).

[0148] The F protein sequence of the ON1 strain was selected based on amino acids 1-513 of the extracellular region of the ON1 strain F0 protein (Genebank: QYW11910) (SEQ ID NO:4). The C-terminus and T4-foldon were linked by the tetrapeptide linker SAIG ​​(SEQ ID NO:38). The pep27 peptide and part of the fusion peptide were removed from F1 and F2, and they were linked by the linker SC linker (positions 109-137 of the native F0 protein were replaced by the SC linker: GGSGSGRS (SEQ ID NO:47)), named ON1-RSV-F(SC).

[0149] V is a key neutralizing epitope. Based on the existing structural analysis of the RSV F protein (PDB ID: 4JHW), 11 residue sites located in or near the allosteric region RR1 were selected for mutation. The percentage of hydrophobic side chain exposure area was calculated using Discovery Studio 2013 software, and the results are shown in Table 2. The hydrophobic side chain exposure area of ​​these sites is less than 50%, so they are relatively embedded. Among them, two sites are located in The epitope region included 6 sites in the V epitope region, 2 sites in the III epitope region, and 1 site in the IV epitope region. Saturation mutations were then designed for each site, and the binding affinity of each mutant to different epitope antibodies was evaluated using ELISA.

[0150] Table 2. Percentage of exposed area of ​​hydrophobic side chains of amino acids

[0151] Experimental Results: Single clones of the saturated mutant library were expressed using the clean protein in 96-well plates. The binding value between the mutant protein and the antibody was detected at OD450 nm. Mutants with binding values ​​were sequenced to confirm the mutation site and type. Specific results are shown in Table 3.

[0152] Table 3

[0153] Based on A2-RSV-F(SC-M1), mutation sites S55A, V185E and S405V with enhanced binding values ​​were introduced to construct mutants as shown in Table 4 below.

[0154] Table 4 RSV F combinatorial mutation design

[0155] Results: The combined mutants were expressed and purified in 293E. The protein was diluted to the same concentration (20 ng / mL), and the binding ability between the mutants and antibodies was detected by ELISA. The yield of the combined mutants M2-M6 was 90-260 mg / L. The binding value of the control A2-RSV-F (SC-M1) was defined as 100%. The relative binding values ​​of other mutants were higher than those of A2-RSV-F (SC-M1), indicating that the combined mutants can enhance the binding affinity to antibodies. Binding of epitope antibodies (Table 5).

[0156] Table 5

[0157] Based on the modifications of M1, M3, and M6, mutation sites I206, N227, L138, and M97 were introduced to obtain a series of mutants (Table 6).

[0158] Table 6 Mutation Modification Design

[0159] Experimental Results: The stability of the designed RSV F protein mutant was evaluated using a stress test. During the heat shock test, the supernatant of the designed mutant was incubated at 60°C for 20 min and analyzed using pre-fusion assays in an ELISA kit. Epitope-specific monoclonal antibodies D25, AM22, RSD5, and pre-fusion trimer-specific antibody AM14 were detected. The ratio of antibody reactivity in stressed samples to that in unstressed samples was defined as the stress resistance parameter. More stable mutants were expected to have higher stress resistance. During the pH tolerance assay at room temperature, the reactivity of pre-fusion antibodies in the supernatant incubated at pH 2.5 for 1 h was compared with that in the fresh culture supernatant. The activity ratio was defined as the mutant's low pH tolerance. More stable mutants were expected to have higher tolerance to low pH.

[0160] The antibody binding values ​​of the combined mutants were measured after expression in the supernatant of 96-well plates. The supernatant was diluted 4000-fold and heat-shocked at 60℃ for 20 min to obtain the heat-shock samples. The binding values ​​of the supernatant and each epitope antibody before and after heat shock were detected by ELISA. The results are shown in Table 7 below. After treatment at 60℃ for 20 min, the heat shock resistance of each epitope of mutants A2-F(SC-M43), A2-F(SC-M44), A2-F(SC-M45), and A2-F(SC-M46) was significantly better than that of the control A2-F(SC-M1). In particular, mutant A2-F(SC-M44) showed the best heat shock resistance.

[0161] Table 7 Results of RSV preF stability mutation point screening

[0162] 1.2 RSV preF protein V epitope library construction, screening, and optimal site reversal mutation

[0163] Library construction was conducted by randomly mutagenesis of the key epitope region V. The selected amino acid regions for library construction were: 149-ASGVAVS-155, 298-AYVVQLP-304, 53-YTSVITI-59, and 187-VLTSKVL-193, for a total of four regions. Random mutations were performed at each site. The harvested supernatant was heat-shocked at 60℃ for 20 min, and the antibody binding value was detected by ELISA. Clones with high binding values ​​after heat shock were sequenced. The optimal mutation sites S55A, S55V, and S190V were obtained. These mutation sites were combined with or replaced with mutation sites in A2-F(SC-M44)(S55A / V185E / N227T / S405V) (SEQ ID NO:19). The mutation sites were introduced into the ON1 strain (SEQ ID NO:4) and the BA9 strain, respectively, to obtain the following mutants. The design scheme is detailed in Table 8.

[0164] Table 8 RSV F combined mutation scheme design

[0165] Experimental results: The binding ability of each mutant to different antibodies was detected by ELISA. The results are shown in Table 9.

[0166] Table 9

[0167] 1.3 Modification of RSV preF protein p27 linker

[0168] Based on BA9-RSV-F(SC-M65) (containing the following mutations: S55V / S190V / N227T / S405V), the linker type and length between F1 and F2 peptides were optimized, and different types of linkers were designed, that is, different F2 peptides (the C-terminus of the F2 peptides are PAVN) were linked through different linkers. 104 PAV 103 PA 102 or P 101 The specific schemes for F2 and F1 peptides are shown in Table 10. A similar method was also used to link F2 and F1 peptides based on A2-RSV-F (SC-M65) and ON1-RSV-F (SC-M65) (Table 12).

[0169] Table 10 F2 / F1 Linker Modification

[0170] The detection results are shown in Table 11. The supernatant from cells expressing 293 was heat-treated at 60℃ for 20 min, and the results were then analyzed by ELISA to detect the expression of each mutant. The binding of the epitope (D25+AM22+RSD5) antibody at OD450nm was analyzed. The OD values ​​were sorted from high to low, with BA9-RSV-F (SC-M65) as a control. Subsequently, some linkers were selected to perform universality verification on different strains.

[0171] Table 11 ELISA detection results with different linker designs

[0172] Three types of linkers, SC11, SC12 and SC24, were selected and constructed onto different strains (Table 12).

[0173] Table 12 Linker design schemes for different strains

[0174] Results: Different mutants were detected by ELISA after a dilution of 3200x, and post epitopes were detected by ELISA after a dilution of 10x. The three forms of linker, SC11, SC12 and SC24, all showed good heat shock resistance and low pH tolerance in different strains (Table 13).

[0175] Table 13 ELISA detection results for different linker-modified strains

[0176] 1.4 Exemplary RSV F protein mutants containing combined mutations of SC11 linker and S290G

[0177] To enhance the binding force of the S155 and S290 sites, saturation mutations (20 × 20) were performed on these two sites. The superior mutation site S290G was obtained by ELISA screening of the supernatant. Based on SC11-M65, the mutation site S290G was introduced, thus constructing the SC11-M83 mutant (Table 14). For both SC11-M65 and SC11-M83, positions 105-144 of the native protein were replaced with SC11 linker:SGS.

[0178] Table 14 contains exemplary RSV F protein mutants with combined mutations of the SC11 linker and S290G.

[0179] The test results are as follows: Introducing the S290G mutation site can increase the resistance of the F protein mutant to... The epitope, the trimer epitope, and the heat shock resistance and low pH 2.5 tolerance of the protein, along with the lower postF protein conformation at the same dilution (Table 15).

[0180] Table 15 Combined mutations of SC11 linker and S290G

[0181] 1.5 Analysis of the contribution of RSV preF preferred molecular mutation points

[0182] After testing the stability of the prefusion conformation under the aforementioned immunogen heat shock or acid treatment conditions, the mutant molecule SC11-M83 was obtained. To verify the contribution of each mutation site to the stability of the preF conformation, a single-point reverse mutation method was used, and the stability of the preF conformation in the supernatant was verified. The results showed that the five introduced mutation sites all played a role in improving the heat shock resistance of the preF conformation or reducing the postF content (Figures 1 and 2).

[0183] Example 2: RSV preF protein structure characterization

[0184] A2-RSV-F (SC11-M83) was named A2F protein (SEQ ID NO:10), and BA9-RSV-F (SC11-M83) was named BA9F protein (SEQ ID NO:22). The structure of the trimer was confirmed by high performance liquid size exclusion chromatography (SEC-HPLC), dynamic light scattering DLS, and negative staining electron microscopy.

[0185] A2F or BA9F proteins are homotrimeric proteins with a prefusion conformation formed by three identical single strands linked by non-covalent bonds. The theoretical molecular weight of each sugar-free single strand is approximately 53 kDa, and the theoretical molecular weight of the sugar-free trimer is approximately 160 kDa. Each single strand contains three theoretical N-linked glycosylation sites. Glycosylation modification usually causes changes in the apparent molecular weight of the protein.

[0186] SEC-HPLC showed that the purity of A2F protein (Figure 3A) and BA9F protein (Figure 3B) was greater than 98%, and the average apparent molecular weight was about 160 kDa, which was basically consistent with the theoretical molecular weight (Figure 3). DLS detection showed that the trimer molecular diameter of A2F protein and BA9F protein was ~10 nm, which was consistent with the reported preF particle size of ~11 nm (Killikelly, AM, et al. 2016) (Table 16).

[0187] Table 16. Particle size and distribution of A2F and BA9F trimer proteins

[0188] Transmission negative staining electron microscopy was used to examine the complexes formed by A2F and BA9F proteins with D25-Fab (Shenzhou Cell) that specifically recognize the pre-conformation F protein. The preF-D25 and preF-AM14 complexes (specifically recognizing the pre-conformation F protein trimeric epitope) were analyzed. Particles of different morphologies from the preF-D25 and preF-AM14 complexes were extracted from micrographs and classified in two dimensions (2D class). Results showed that the A2F and BA9F protein trimers primarily formed two D25 copies with D25-Fab, binding to the top of the preF protein, and primarily formed three AM14 copies with AM14-Fab, binding to the middle of the preF protein. Electron microscopy further confirmed that the preF trimers in both A2F and BA9F proteins existed in a relatively stable pre-fusion conformation, consistent with the previously reported RSV preF+D25-Fab / AM14-Fab complex conformation (McLellan, JS, et al. 2013; Harshbarger, W., et al. 2021).

[0189] Example 3: Stability of RSV F protein mutant

[0190] 3.1 Freeze-thaw stability

[0191] The stock solutions of A2F (A2-RSV-F(SC11-M83)) and BA9F (BA9-RSV-F(SC11-M83)) were stored at -25°C for at least overnight. The samples were then removed and allowed to thaw naturally at room temperature. After complete thawing, the samples were placed back at -25°C. This process was repeated four times, and samples were taken at 0 and 4 cycles to detect the relative binding activity and purity of the mutants (SEC-HPLC, Agilent).

[0192] Relative binding activity detection method: Indirect enzyme-linked immunosorbent assay (ELISA) is used. Coated antibodies (AM14, D25, and 9501) are bound to a solid-phase carrier to form a solid-phase antibody. Unbound antibodies and impurities are washed away. Serial concentrations of A2F (or BA9F) reference standard and the test sample are added to bind with the solid-phase antibody, forming a solid-phase antigen-antibody complex. Then, horseradish peroxidase-labeled detection antibody (anti-T4 monoclonal antibody R124 / HRP) is added. A2F and BA9F on the solid-phase immune complex bind to the enzyme-labeled antibody. The substrate is added, and the enzyme on the solid phase catalyzes the substrate to form a colored product. After terminating the reaction, the absorbance value at 450 nm is read using an ELISA reader. The sample concentration is plotted on the x-axis, and the average absorbance intensity (OD450) is plotted on the y-axis. 样品 -OD450 空白对照 Using y as the ordinate, the EC values ​​of the control standard and the stability test sample are obtained. 50 EC values ​​were calculated for the control standard and the stability test samples. 50 The ratio was used to examine the relative binding activity of A2F and BA9F.

[0193] The A2F and BA9F proteins were repeatedly frozen and thawed four times at temperatures below -25°C. The relative activity and purity did not change significantly, indicating that the modified proteins have good freeze-thaw stability (Table 17).

[0194] Table 17 Freeze-thaw stability results of A2F and BA9F proteins

[0195] 3.2 Accelerated stability test (2–8℃)

[0196] A2F and BA9F were placed at 2–8°C, and samples were taken at 0, 1, and 3 months, respectively. The relative binding activity and purity (SEC-HPLC, manufacturer: Agilent) of the samples were detected. The specific detection method was the same as that for freeze-thaw stability.

[0197] The results of the accelerated stability study are shown in Table 18. When A2F and BA9F were stored at 2–8°C for 3 months, their relative activity and purity did not change significantly, indicating that the modified protein has good accelerated stability.

[0198] Table 18 Results of accelerated stability studies of A2F and BA9F at 2–8℃

[0199] 3.3 Long-term stability study (below -25℃)

[0200] A2F and BA9F were stored at -25°C or below, and samples were taken at 0 and 3 months, respectively. The relative binding activity, specific activity, and purity (SEC-HPLC) of the samples were then analyzed. The specific testing methods were the same as for freeze-thaw stability.

[0201] The results of the long-term stability study are shown in Table 19. When A2F and BA9F were stored at -25℃, their relative activity and purity did not change significantly, indicating that the modified protein has good long-term stability.

[0202] Table 19 Results of stability study of A2F and BA9F at -25℃

[0203] Example 4: RSV preF protein binding activity with neutralizing antibody

[0204] The method for detecting preF protein binding activity is as follows:

[0205] Dilute D25, AM14, and 4D7 antibodies to 2 μg / mL and coat each well with 100 μL of the solution in a 96-well plate. Incubate overnight at 2-8°C. The next day, wash the plate and block at room temperature for at least 1 hour. Add 100 μL of different concentrations of BA9F, A2F, and Arexvy (GlaxoSmithKline Biologicals) and Abrysvo (Pfizer Pharmaceuticals Ltd) to each well and incubate for 1-2 hours. Perform experiments in duplicate wells. After washing to remove unbound samples, add 1 μg / mL of biotin-labeled anti-T4-Flodon detection antibody T4-Foldon-R124-biotin (Shenzhou Cell) and incubate. Wash the plate three times. Then add horseradish peroxidase-labeled streptavidin-HRP and incubate for 1 hour, followed by five washes. Finally, add substrate chromogenic buffer for color development. Measure OD450 after termination. Plotting protein concentration on the x-axis and OD value on the y-axis, the dose-response curve was analyzed and plotted using GraphPad Prism 8.0 software, and the median effective dose (EC50) was calculated. 50 value.

[0206] The binding activity of A2F protein and BA9F series antibodies recognizing different RSV F protein antigenic epitopes and conformational features was detected by ELISA, and compared with Pfizer Abrysvo vaccine antigen molecules and GSK Arexvy vaccine antigen molecules. The results are shown in Table 20 and Figure 4. Both A2F (A2-RSV-F(SC11-M83)) and BA9F (BA9-RSV-F(SC11-M83)) trimers can bind with high affinity to neutralizing antibodies D25 (Figure 4A), AM14 (Figure 4B), and EC. 50 The binding concentrations ranged from 6.7 to 8.6 ng / mL, and their binding abilities were higher than those of GSK Arexvy and Pfizer Abrysvo vaccine antigens. Furthermore, the A2F and BA9F proteins showed virtually no binding to 4D7 (epitope I, specifically recognizing the post-conformation) (Figure 4C), and their binding abilities were lower than those of GSK Arexvy and Pfizer Abrysvo, indicating that these two proteins have a lower post-F conformation. The binding of neutralizing antibodies suggests that both A2F and BA9F proteins possess complete biological functions and structural basis for inducing immunogenicity, and are proteins with low levels of post-conformation proteins.

[0207] Table 20. Neutralizing antibody binding to ECs at different epitopes of A2F and BA9F proteins. 50

[0208] Example 5: Validation of the immunogenicity of RSV preF protein

[0209] exist The immunogenicity of the RSV pre-conformation modified F protein and the post-conformation F protein were compared in Balb / c mice. The RSV preF protein was the preferred molecule A2F (A2-RSV-F(SC11-M83)), while the A2-RSV-F-post protein was the A2 strain F protein with a post conformation designed according to the literature (Swanson, KA, et al. 2011). The postF protein was obtained by fusing amino acids 1-136 and 146-524 of the extracellular region of the F protein. Compared with the extracellular region sequence of the wild-type RSV F protein, the N-terminal part of the fusion peptide was missing amino acids 137-145.

[0210] 5.1 The preF protein can induce higher antibody titers that bind to preF.

[0211] Female Balb / c mice aged approximately 6-7 weeks were immunized with 1.5 μg, 3.0 μg, or 6.0 μg of preF or postF protein, respectively, for a total of two immunizations, 14 days apart. Serum samples were collected 14 days after the second immunization for the determination of antigen IgG titer, neutralizing titer, and serum antibody levels competing for different epitopes.

[0212] Experimental grouping and immunization: The mice were immunized with the corresponding vaccine samples as shown in the table below (Table 21). The immunization date, immunization method, immunization sample name and dosage were recorded. There were 5 mice in each group.

[0213] Table 21

[0214] *SCT-VA02B is an adjuvant solution (MF59) (Shenzhou Cell).

[0215] Animal immunization, blood collection

[0216] Balb / c mice were immunized with 100 μL of protein vaccine via intramuscular injection in one hind limb on days 0 and 14. On day 14 post-immunization, approximately 150 μL of blood was collected from the orbital sinus of the mice, and the serum was used to determine the antigen IgG titer and neutralization titer.

[0217] Mouse serum antibody titer detection

[0218] Coat 100 μL of 5 μg / mL A2F(preF) (Shenzhou Cell) or A2-RSV-F-post(postF) (Shenzhou Cell) stock protein solution onto 96-well ELISA plates, incubating overnight at 2-8°C. After washing and drying the ELISA plates, add 300 μL of 2% BSA blocking buffer per well and block at room temperature for at least 1 hour. Serially dilute immunized mouse serum for testing, and use unimmunized mouse serum diluted to the same concentration as negative control serum. Add 100 μL of the sample to the corresponding antigen-coated ELISA plate and incubate at room temperature for approximately 1-2 hours, washing 3 times. Add 100 μL of 80 ng / mL rabbit anti-mouse IgG F(ab)2 / HRP secondary antibody per well, incubating at room temperature for approximately 1 hour, washing 5 times. Add substrate chromogenic buffer for color development, stopping with 2M H2SO4, and read the OD value using an ELISA reader. 450 Using Microsoft Office Excel, the OD values ​​of negative control serum at various dilutions were analyzed. 450 The average value is taken as NC, and the method threshold is 2.1*NC. The maximum dilution factor greater than this threshold is reported as the antibody titer.

[0219] Experimental results

[0220] Under three immunization dose conditions, the ratio of anti-preF antibody titer to anti-postF antibody titer in preF protein-immunized serum was 4.00–4.59, while the ratio in postF protein-immunized serum was only 0.07–0.09 (Figure 5), indicating a significant conformational difference in antibody titers induced by the pre and post conformations of RSV F protein. These results demonstrate that the preF protein-induced antibody titer against the preF conformation is significantly higher than that against the postF protein.

[0221] 5.2 preF can induce higher neutralizing antibody titers and antibody levels targeting different neutralizing epitopes.

[0222] The specific neutralizing antibodies against respiratory syncytial virus (RSV) in the serum of Balb / c mice were detected using the 50% endpoint method.

[0223] Experimental methods

[0224] The specific neutralizing antibody against respiratory syncytial virus (RSV) in Balb / c mouse serum was detected using a 50% endpoint method. 50 μL / well of mouse immune serum at different dilutions (immunization method as described in Example 5.1) was added to a 96-well plate, followed by 50 μL / well of 25 TCID50 solution. 50 RSV A2 strain virus was mixed and incubated at 37°C with 5% CO2 for 2 hours. After incubation, 100 μL / well was inoculated with 1×10⁻⁶ cells / well. 4 Hep 2 cells were used, with negative control wells (cells only) and positive control wells (cells and virus). After mixing, the cells were incubated statically at 37°C and 5% CO2 for approximately 5 days. After incubation, CPE was observed under a microscope, and the number of wells showing the same cell morphology as the negative control group (i.e., the number of wells without CPE) was counted. The IC50 was calculated using the Karber formula. 50 That is, lgLd50 = L + d(S - 0.5), where L is the logarithm of the lowest dilution of the sample, d is the class interval, and S is the number of non-CPE wells / the number of sample replicates. 50 =10^-lgLd50, which is the neutralizing antibody titer (NAT). 50 GrapPad Prism software is used for data statistics.

[0225] Experimental results

[0226] Figure 6 shows the neutralizing antibody titers against RSV A2 virus strain in the serum of mice immunized with RSV preF and postF proteins under three immunization doses. The neutralizing antibody titers induced by A2F (preF) protein were 3.2–5.7 times higher than those induced by postF protein, indicating that A2F (preF) protein can induce higher neutralizing titers. These results suggest that the stability-optimized F trimer protein can induce higher neutralizing titers after locking the pre conformation.

[0227] The above results indicate that, compared to the post-conformation F protein, the stability-optimized pre-conformation F trimer protein (A2F) can bind to pre-specific neutralizing antibodies with high affinity, induce higher pre-F conformation antibody titers, and achieve higher specificity against pre-specific neutralizing antibodies. The antibody content and neutralizing titer of the V epitope.

[0228] sequence list

[0229] References:

[0230] 1. Che, Y., et al. (2023). "Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine." Science Translational Medicine 15(693):eade6422.

[0231] 2. Crowe, JE, et al. (2019). "Alternative conformations of a major antigenic site on RSV F." PLOS Pathogens 15(7):e1007944.

[0232] 3.Flynn,J.A.,et al.(2016)."Stability characterization of a vaccine antigen based on the respiratory syncytial virus fusion glycoprotein."PLoS One 11(10):e0164789.

[0233] 4.Graham,B.S.(2017)."Vaccine development for respiratory syncytial virus."Curr Opin Virol 23:107-112.5.Harshbarger,W.,et al.(2021)."Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein."mAbs 13(1).

[0234] 6.Killikelly,A.M.,et al.(2016)."Pre-fusion F is absent on the surface of formalin-inactivated respiratory syncytial virus."Scientific Reports 6(1).

[0235] 7.Mantis,N.J.,et al.(2016)."Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein."Plos One 11(10):e0164789.

[0236] 8.McLellan,J.S.,et al.(2013)."Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody."Science 340(6136):1113-1117.

[0237] 9.Ngwuta,J.O.,et al.(2015)."Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera."Sci Transl Med 7(309):309ra162.

[0238] 10.Swanson,K.A.,et al.(2011)."Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein(RSV F)to elicit high neutralizing antibody titers."Proceedings of the National Academy of Sciences 108(23):9619-9624.

[0239] 11.Gilman,M.S.A..,et al.(2015)."Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary,Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein."PLOS Pathogens 11(7):e1005035.

Claims

A mutant of the recombinant RSV F protein contains two polypeptides, F2 and F1, from the N-terminus to the C-terminus. The mutant contains the following mutations relative to the wild-type RSV F protein sequence: 55V (preferred) / A, 227T (preferred) / L / I / M, and 405V; preferably, the mutant also contains 185E or 190V (preferred); more preferably, the mutant also contains 290G; the RSV F position corresponds to the amino acid sequence of the reference RSV F0 polypeptide shown in SEQ ID NO:1; wherein Optionally, The mutant does not contain the pep27 peptide, and (1) The F2 polypeptide is directly linked to the F1 polypeptide, or (2) The F2 polypeptide and the F1 polypeptide are linked by a heteropeptide linker. Preferably, the linker is selected from: SGS (preferred), SSG, SPS, SGSG (SEQ ID NO:42), SGGS (SEQ ID NO:41), SGGG (SEQ ID NO:45), SGG, S, GSP, GSGS (SEQ ID NO:43), GSG, GSAS (SEQ ID NO:40), GS, GGSGG (SEQ ID NO:44), GGSG (SEQ ID NO:46), GGS, GGGS (SEQ ID NO:39) or G; Optionally, The F2 and F1 polypeptides respectively contain positions 26-104 or 26-108 of the RSV F protein, and positions 145-513 or 138-513. More preferably, the sequence of the F2 polypeptide is as shown in SEQ ID NOs:11, 14, 17, 20, 23, 26, 29 or 32, or the sequence of the F2 polypeptide has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the sequence shown in SEQ ID NOs:11, 14, 17, 20, 23, 26, 29 or 32, provided that the F2 polypeptide contains the following mutation: S55V / A; and / or The F1 polypeptide sequence is as shown in SEQ ID NOs:12, 15, 18, 21, 24, 27, 30 or 33, or the F1 polypeptide sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the sequence shown in SEQ ID NOs:12, 15, 18, 21, 24, 27, 30 or 33, provided that the F1 polypeptide contains the following mutations: N227T / L / I / M and S405V, preferably also containing V185E or S190V, more preferably also containing S290G. The mutant of claim 1, wherein the mutant comprises the following mutations relative to the wild-type RSV F protein sequence: S55V (preferred) / A, N227T (preferred) / L / I / M and S405V, Preferably, the mutant further comprises V185E or S190V (preferred). More preferably, the mutant further comprises S290G. The mutant as described in claim 1 or 2, wherein The mutant does not contain a furin restriction site, and / or The F1 polypeptide does not contain a transmembrane domain or an intracellular domain. The mutant of any one of claims 1-3, wherein the mutant further comprises a signal peptide. Preferably, the signal peptide sequence is selected from the sequence shown in SEQ ID NOs:34-36. The mutant of any one of claims 1-4, wherein the C-terminus of the F1 polypeptide is linked to a polymerizing domain (preferably a trimerizing domain); Preferably, the polymerized domain connected to the C-terminus comprises a T4-foldon trimerized domain; and / or Preferably, the T4-foldon trimerization domain comprises the amino acid sequence shown in SEQ ID NO:37; and / or Preferably, the polymerizing domain (such as the T4-foldon trimerizing domain) is linked to amino acid residue 513 of the RSV F protein mutant; and / or Preferably, the multimerizing domain (such as the T4-foldon trimerizing domain) is directly or via a peptide linker (such as SAIG ​​(SEQ ID NO:38)) connected to the F1 polypeptide. The mutant of any one of claims 1-5, compared with the corresponding wild-type RSV F protein, exhibits increased stability under heat treatment at 60°C for 20 min, wherein said stability is associated with specific recognition by said mutant. The binding of epitopes to antibodies (such as combined antibodies against D25, AM22, and / or RSD5) is measured. The mutant of any one of claims 1-6 has increased stability at pH 2.5 for 20 min compared to the corresponding wild-type RSV F protein, wherein the stability is measured by the binding of the mutant to a preF-specific antibody (such as a combination antibody of D25, AM22 and / or RSD5). The mutant of any one of claims 1-7, wherein, when the same dose of RSV F antigenic peptide is administered to a subject, the humoral and / or cellular immune responses induced by the mutant are comparable to or higher than those induced by wild-type RSV F protein; Preferably, the induced humoral immune response is increased by at least 3-6 times. The mutant of any one of claims 1-8, wherein the mutant comprises The sequence shown in SEQ ID NO:10 or SEQ ID NO:22, or The sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:10 or SEQ ID NO:22, provided that the mutant contains the following mutations: S55V, N227T, and S405V, preferably also containing S190V, and more preferably also containing S290G. The mutant of any one of claims 1-9, wherein the wild-type RSV is subtype A or subtype B. The mutant according to any one of claims 1-10 is in the form of a trimer. Nucleic acid molecules that encode the mutants as described in any one of claims 1 to 11, Preferably, the nucleic acid molecule is mRNA or DNA. More preferably, the nucleic acid molecule is mRNA. A carrier comprising the nucleic acid molecule of claim 12. A host cell comprising the nucleic acid molecule of claim 12 or the vector of claim 13. An immunogenic composition comprising the recombinant RSV F protein mutant of any one of claims 1-11, the nucleic acid molecule of claim 12, or the vector of claim 13. The immunogenic composition of claim 15 further comprises an adjuvant. The immunogenic composition of claim 16, wherein the adjuvant is selected from Th1 adjuvants or aluminum adjuvants; Preferably, the Th1 adjuvant is selected from: (1) Oil-in-water emulsion adjuvants, such as (a) MF59; (b) SAF; or (c) Ribi™ adjuvant system (RAS); (2) Saponin adjuvants, such as Quil A or QS-21; (3) Bacterial lipopolysaccharides, such as AGP; (4) Synthesize polynucleotides, such as oligonucleotides containing CpG motifs; (5) Cytokines, such as interleukins, interferons, GM-CSF, M-CSF, TNF, or co-stimulatory molecules B7-1 and B7-2, The interleukin is preferably IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, or IL-18, and the interferon is preferably gamma interferon; (6) Mucosal adjuvants; and (7) Other substances that act as immunostimulants to enhance the effectiveness of the composition; Preferably, the aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc. The immunogenic composition according to any one of claims 15-17 is a liquid or lyophilized product. A vaccine comprising any one of claims 1-11, a recombinant RSV F protein mutant, the nucleic acid molecule of claim 12, the vector of claim 13, or the immunogenic composition of any one of claims 15-18. A method for treating or preventing diseases or symptoms caused by RSV using a mutant of the recombinant RSV F protein of any one of claims 1-11, a nucleic acid molecule of claim 12, a vector of claim 13, an immunogenic composition of any one of claims 15-18, or a vaccine of claim 19. The mutant of the recombinant RSV F protein of any one of claims 1-11, the nucleic acid molecule of claim 12, the vector of claim 13, the immunogenic composition of any one of claims 15-18, or the vaccine of claim 19, are used to treat or prevent diseases or symptoms caused by RSV.

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