CN117586358A - Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity - Google Patents

Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity Download PDF

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CN117586358A
CN117586358A CN202410079890.3A CN202410079890A CN117586358A CN 117586358 A CN117586358 A CN 117586358A CN 202410079890 A CN202410079890 A CN 202410079890A CN 117586358 A CN117586358 A CN 117586358A
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protein
polypeptide
domain
rsv
recombinant
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CN117586358B (en
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胡颖嵩
邓家荔
张元杰
任朵朵
李菡
杨克俭
洪坤学
刘勇
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Jiangsu Ruike Biotechnology Co ltd
Wuhan Ruike Biotechnology Co ltd
Abzymo Biosciences Co ltd
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Wuhan Ruike Biotechnology Co ltd
Abzymo Biosciences Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
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    • A61P31/14—Antivirals for RNA viruses
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

The invention provides an immunogenic Respiratory Syncytial Virus (RSV) polypeptide, and relates to the technical field of biological medicines. The antigen and the vaccine prepared by the antigen have high expression quantity, good stability and strong immunogenicity.

Description

Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity
Technical Field
The application belongs to the technical field of biological medicine, and particularly relates to an immunogenic Respiratory Syncytial Virus (RSV) polypeptide, a preparation method thereof and application thereof in preparing vaccine for preventing RSV infection.
Background
Respiratory viral infections are one of the most important public health burdens worldwide, resulting in millions of hospitalizations worldwide each year. Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is the leading cause of acute lower respiratory tract infection in children under the age of 2 and adults over the age of 65. Although the respiratory tract disease caused by RSV infection is severely compromised, no effective therapeutic and prophylactic vaccine specific to RSV is currently available. The major direction of development of RSV vaccines worldwide into clinical stages has focused on live attenuated vaccines, recombinant subunit vaccines and viral vector vaccines. RSV vaccine development is mainly directed to 3 target populations, including infants, pregnant women, and the elderly (no less than 60 years). Infants older than 6 months, either achieved passive immune protection mainly by mAbs, or achieved passive protection by placental transfer of antibodies after vaccination with RSV vaccine by pregnant women; infants older than 6 months of age can be protected by active immunization with a live attenuated RSV vaccine; the development of RSV vaccines for pregnant women and the elderly is based on subunit vaccines.
The choice of mainstream vaccine antigens is mainly focused on F proteins, especially F proteins in pre-fusion conformation (PreF). The F protein in the pre-fusion conformation may expose more potential epitopes with neutralizing activity than the F protein in the post-fusion conformation, such as epitope Φ and epitope V only present in PreF. Antibodies generated against the epitope phi and the epitope V have excellent neutralizing activity, and after being combined with F protein in a pre-fusion conformation, the antibodies can block virus membrane fusion mediated by F protein conformational change. Despite extensive research on the conformational stability of RSV F protein prior to fusion, the safety and efficacy of its use as a vaccine remains a significant challenge.
Accordingly, it is an urgent need for a vaccine comprising the same to provide an RSV antigen having high safety and enhanced immunogenicity and improved stability.
Disclosure of Invention
To solve the above technical problems, the present invention provides a Respiratory Syncytial Virus (RSV) polypeptide having immunogenicity.
In one aspect, the invention discloses an immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising a recombinant ectodomain of the RSV F protein, corresponding to amino acid residues 26-513 of the wild-type RSV F protein sequence depicted in SEQ ID NO. 1, wherein the recombinant ectodomain comprises a pair of disulfide mutations V144C+I407C, S C+N460C or A149C+Y458C.
The recombinant extracellular domain in the technical scheme of the invention further comprises two other pairs of disulfide bond mutations, wherein one mutation site in one pair is at 137 to 216 sites of the F protein, and one mutation site in the other pair is at 460 to 500 sites of the F protein.
In the technical scheme of the invention, the pair of disulfide bond mutations of the mutation site at 137 to 216 positions of F protein refers to I167C+Y 286C, S C+L188C, H159 15C+I291C or G151C+I288C.
The mutation of disulfide bonds of the mutation site at 460 to 500 positions of the F protein in the technical scheme of the invention refers to K399C+S485C, S443C+S466C, T C+I475C, I C+P480C or F387C+I492C.
The invention discloses an immunogenic Respiratory Syncytial Virus (RSV) polypeptide, comprising an RSV F protein recombinant ectodomain, corresponding to amino acid residues 26-513 of the wild type RSV F protein sequence shown in SEQ ID NO. 1, wherein the recombinant ectodomain comprises the following combination of disulfide mutations:
A. a pair of disulfide mutations selected from V144C + I407C, S146C + N460C or a149C + Y458C;
B. a pair of disulfide mutations in positions 137 to 216 of the F protein at one mutation site; and
C. one of the mutation sites is a pair of disulfide mutations at positions 460 to 500 of the F protein.
In the technical scheme of the invention, the pair of disulfide bond mutations of the mutation site at 137 to 216 positions of F protein refers to I167C+Y 286C, S C+L188C, H159 15C+I291C or G151C+I288C; wherein the pair of disulfide mutations at positions 460 to 500 of the F protein at the one mutation site refers to K399C+S485C, S C+S466C, T323C+I475C, I C+P480C or F387C+I492C.
The polypeptide of the technical scheme of the invention, wherein the recombinant ectodomain comprises one of the following disulfide mutation combination schemes:
A. V144C + I407C, I C + Y286C and F387C + I492C;
B. V144C + I407C, I C + Y286C, S C + L188C and F387C + I492C.
The polypeptide of the technical scheme of the invention, wherein the recombinant ectodomain further comprises a cavity filling mutation N67F or N67F+S190V+V296F+V495L.
The polypeptide in the technical scheme of the invention, wherein the recombinant extracellular domain further comprises mutation P102A+I379V+M V, S46G or G184N for increasing the expression level.
The polypeptide in the technical scheme of the invention, wherein the recombinant ectodomain comprises a recombinant F2 domain at least comprising amino acid residues 26-103 of RSV F protein and a recombinant F1 domain at least comprising amino acid residues 145-513 of RSV F protein.
The recombinant ectodomain described in the present invention comprises an epitope that is present only in the pre-fusion conformation, said epitope comprising residues 62-69 and 196-209 of the wild-type RSV F protein sequence depicted in SEQ ID NO. 1.
The polypeptide in the technical scheme of the invention, wherein the recombinant F2 domain is directly connected with the recombinant F1 domain or is connected with the recombinant F1 domain through a linker.
In some embodiments, wherein the recombinant F2 domain is linked to the recombinant F1 domain by linker means that the C-terminus of the F2 domain and the N-terminus of the F1 domain are substituted with linker selected from GGPGGS, GAPEPGE or GGSGGSG.
In some embodiments, wherein the C-terminus of the polypeptide comprises a multimerization domain.
In some embodiments, wherein the multimerization domain is a trimerization domain or a domain that can achieve more aggregation.
In some embodiments, wherein the multimerization domain is located at the C-terminus of the recombinant ectodomain, directly linked to the C-terminus of the recombinant ectodomain or linked via a linker sequence.
In some embodiments, optionally, the linker sequence is a GGGSSGS, GSGSG or G and S composed 4-10 amino acid sequence.
In some embodiments, wherein the multimerization domain is a trimerization domain, the trimerization domain promotes the formation of a trimer of 3F 1/F2 heterodimers.
In some embodiments, wherein the multimerization domain is a domain that can achieve more multimerization, e.g., ferritin that can self-assemble to form a twenty-four mer.
In some embodiments, the multimerization domain is preferably a foldon domain or a ferritin domain.
In the present application, "ferritin" refers to noctuid ferritin or helicobacter pylori ferritin. In the present application, H.pylori ferritin has a single-stranded structure. In the present application, the ferritin may be a mutant of spodoptera exigua or helicobacter pylori ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids. Illustratively, the ferritin amino acid sequence used herein is ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS.
The invention also discloses application of the polypeptide in preparing a vaccine for preventing RSV infection.
In some embodiments, wherein the vaccine provides protection against infection by at least one of RSV subtype a and/or subtype B.
In some embodiments, the vaccine for preventing RSV infection comprises an adjuvant in the form of liposomes.
In some embodiments, the liposomal adjuvant comprises components of MPL, QS-21, and cholesterol.
The polypeptides of the invention comprise a multimerizing (e.g., trimerizing) domain that self-assembles into a twenty-tetramer and a recombinant ectodomain of an RSV F protein, wherein the recombinant ectodomain comprises cavity-filling mutations corresponding to one or more of positions 67, 190, 296, and 495 of the RSV F protein sequence set forth in SEQ ID No. 1.
In some embodiments, wherein the recombinant ectodomain comprises the F2 domain of RSV F protein or a functionally active fragment thereof and the F1 domain or a functionally active fragment thereof.
In some embodiments, the F2 domain comprises a RSV F protein polypeptide corresponding to amino acids 26-105 of SEQ ID NO. 1, and the F1 domain comprises a RSV F protein polypeptide corresponding to amino acids 137-516 of SEQ ID NO. 1.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising at least amino acid residues 26-105 of the RSV F protein and a recombinant F1 domain comprising at least amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising amino acid residues 26-105 of the RSV F protein and a recombinant F1 domain comprising amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant F2 domain and the recombinant F1 domain are linked by a linker sequence.
In some embodiments, wherein the recombinant F2 domain and the recombinant F1 domain are linked by a GGGSSGS, GSGSG, GGPG or G to S composed of a 4-10 amino acid residue linker sequence.
In some embodiments, wherein the G and S constitute a 4-10 amino acid residue linker sequence, optionally GGSGGSG.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising at least amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising at least amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising amino acid residues 147-513 of the RSV F protein.
In some embodiments, wherein the recombinant F2 domain and recombinant F1 domain are linked by a linker sequence of 5-8 amino acid residues consisting of glycine G, proline P, and one or more amino acids selected from serine S, alanine a, glutamic acid E, and lysine K.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, and serine S.
In some embodiments, the linker sequence is a 6 amino acid residue length linker sequence consisting of glycine G, proline P, and serine S, wherein the linker sequence is GGPGGS.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, serine S, and alanine a.
In some embodiments, the linker sequence is a 5 amino acid residue length linker sequence consisting of glycine G, proline P, serine S, and alanine a, wherein the linker sequence is GAPGS.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, alanine a, and glutamic acid E.
In some embodiments, the linker sequence is a 7 amino acid residue length linker sequence consisting of glycine G, proline P, alanine a, and glutamic acid E, wherein the linker sequence is gappge.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, alanine a, glutamic acid E, and lysine K.
In some embodiments, the linker sequence is a 7 amino acid residue length linker sequence consisting of glycine G, proline P, alanine a, glutamic acid E, and lysine K, wherein the linker sequence is gkppapge.
In some embodiments, wherein the cavity filling mutation is selected from one or more of N67F, S190V, V296F, and V495L. In some embodiments, wherein the cavity filling is abrupt to N67F. In some embodiments, wherein the cavity filling mutation is preferably a combination of N67F, S190V, V296F and V495L.
In some embodiments, wherein the substitution that increases expression is selected from p102a+i379v+m V, S46G and/or E218A.
In some embodiments, wherein the recombinant ectodomain further comprises an electrostatic mutation, e.g., in some embodiments the electrostatic mutation is G184N, while in other embodiments, G184N also has an effect of increasing expression.
In some embodiments, disulfide mutations are designed to form intermolecular disulfide bonds (inter disulfide bonds), which are disulfide bonds that link monomers to one another, which can assist in locking the F protein in a pre-fusion conformation, such as v144c+i407C or s150c+y458C, and the like. In some embodiments, the invention contemplates disulfide mutations that are aimed at forming RR1 disulfide bonds capable of immobilizing the distal region of the membrane, said RR1 disulfide bonds being formed by disulfide bond mutation of one of the mutation sites at positions 137 to 216 of the F protein, such as I148c+y286C, S c+l188C, H c+i291C or g151c+i288C. In some embodiments, the invention contemplates disulfide mutations that are aimed at forming RR2 disulfide bonds capable of immobilizing the proximal region of the membrane, said RR2 disulfide bonds being formed by disulfide bond mutation of one of the mutation sites at positions 460 to 500 of the F protein, e.g., k399c+s485C, S443c+s466C, T323c+i475C, I c+p480C or f387c+i492C.
The polypeptides disclosed herein comprise signal peptide sequences required for expression or linker sequences linked to purification tags, which may be removed in the final product.
In some embodiments, the polypeptide further comprises a heterologous signal peptide at the time of construction.
In some embodiments, the heterologous signal peptide is an IgG signal peptide having an amino acid sequence of MGWSCIILFLVATATGVHS.
In some embodiments, the polypeptide also incorporates affinity tags such as strep Tag (amino acid sequence: WSHPQFEK), monoclone Flag-Tag (amino acid sequence: DYKDDDK) and His Tag (amino acid sequence: HHHHHHH) at the time of vector construction, which Tag sequences can be removed in the final product.
In some embodiments, the polypeptides of the invention are soluble trimeric proteins.
In some embodiments, the polypeptide of the invention is a particle in which the tetracosane forms 8 trimeric extracellular domains.
The invention also provides application of the polypeptide in preparing vaccine for preventing RSV infection.
The invention also provides a vaccine for preventing RSV infection, comprising a polypeptide of any of the above; the vaccine provides protection against infection by at least one of the RSV subtypes a and/or B.
The invention discloses a vaccine composition for preventing RSV infection, further comprising an immunoadjuvant comprising at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, cpG, poly (I: C) and QS-21.
In some embodiments, the invention discloses a vaccine for preventing RSV infection comprising an adjuvant in the form of liposomes, the major components of the adjuvant in the form of liposomes comprising MPL, QS-21, DOPC and cholesterol. The dose of the adjuvant may be 0.5ml per dose, containing 50 μg MPL, 50 μg QS-21, 1mg DOPC, 0.25mg cholesterol, 4.835mg sodium chloride, 0.15mg anhydrous disodium hydrogen phosphate, 0.54mg potassium dihydrogen phosphate and water for injection, or may be one half, one quarter or less of the above dose, for example, the main components include 25 μg MPL, 25 μg QS-21, 0.5mg DOPC, 0.125mg cholesterol.
The invention also provides a method for preparing the polypeptide, which comprises the following steps:
s1, synthesizing a DNA sequence corresponding to the polypeptide, and cloning the DNA sequence into a plasmid vector;
s2, transfecting a host cell with a recombinant plasmid vector and expressing the host cell, wherein the host cell is a eukaryotic cell;
s3, purifying the culture product to obtain the polypeptide.
In some embodiments, the vaccines of the present invention generally further comprise a pharmaceutically acceptable carrier and/or excipient such as a buffer. Pharmaceutically acceptable carriers and excipients are well known and may be selected by those skilled in the art. Optionally, the pharmaceutically acceptable carrier or excipient further comprises at least one component that stabilizes solubility and/or stability. Examples of solubilizing/stabilizing agents include detergents, such as laurel sarcosine and/or tween. Other examples of solubilizing agents/stabilizers include arginine, sucrose, trehalose, and the like. Thus, one skilled in the art can select suitable excipients and carriers to produce a formulation suitable for administration to a subject by a selected use. Suitable excipients include, but are not limited to, glycerol, polyethylene glycol, KCl, calcium ions, magnesium ions, manganese ions, zinc ions, and other divalent cation related salts, and the like.
The adjuvant should generally be capable of enhancing a Th 1-biased immune response in a subject or population of subjects receiving vaccine administration and be safe and effective in the subject or population of subjects at the time of selection.
The invention also provides a method for preparing the vaccine for preventing RSV infection, which comprises the following steps:
S1, culturing a mammalian host cell to express the polypeptide;
s2, purifying the polypeptide expressed in the step (1);
s3, fully mixing the purified polypeptide with an immunoadjuvant according to a proportion.
In some embodiments, additional steps are included in gene synthesis, construction of expression vectors, lyophilization of purified proteins, and the like. The expressed fusion polypeptide may be recovered and purified from recombinant cell culture by any one or several of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, filtration, ultrafiltration, centrifugation, anion/cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Finally, high performance liquid chromatography HPLC can be employed in the final purification step.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains RSV polypeptide with high expression quantity and immunogenicity which has specific epitope to Pre-F through the design of stabilizing modification, multimerization (such as trimerization) and the like of wild RSV F protein, and ensures that the RSV polypeptide can cause effective neutralizing antibody reaction and binding antibody reaction to respiratory syncytial virus while maintaining stable Pre-fusion conformation.
Drawings
The following drawings are only for purposes of illustration and explanation of the present invention and are not intended to limit the scope of the invention. Wherein:
fig. 1 (a): molecular sieve analysis results of DS-Cav1A protein supernatant samples in example 3 of the present invention;
fig. 1 (B): molecular sieve analysis results of DS-Cav1B protein supernatant samples in example 3 of the present invention;
fig. 2 (a): electrophoresis results of DS-Cav1A protein supernatant samples in example 3 of the present invention;
fig. 2 (B): electrophoresis results of DS-Cav1B protein supernatant samples in example 3 of the present invention.
Detailed Description
The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, that many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.
Unless defined otherwise, 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 invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
Definition of terms
In the present application, the term "DS-Cav1" is a PreF design concept with reference to CN105473604B, including pre-F trimers stabilized by disulfide bonds between residues 155 and 290 ("DS"), cavity filling mutations S190F and V207L ("Cav 1"), and additional C-terminal fibritin (i.e., foldon) trimerization domains, specific sequences being given in the examples.
In the present application, the term "D25" refers to the antibody described in CN101778866B, the specific amino acid sequence of which is referred to in the specification as SEQ ID NO 1-8.
In the present application, the term "101F" refers to the antibodies described in US11261356, the nucleotide and amino acid sequences of the heavy and light chains of which are referred to in the specification by SEQ ID NOS 3-6.
In the present application, the term "Mota" or "motavizumab" refers to an antibody as described in WO2007002543A2, the specific sequences of which refer to the amino acid sequences SEQ ID NO:48 and SEQ ID NO:11 of the antibodies A4B4L1FR-S28R (aka motavizumab) in the patent specification.
In this application, each mutant of the present invention was designed and prepared based on the amino acid sequence of the wild-type (abbreviated as WT) RSV F protein described in SEQ ID NO. 1. The F2 polypeptide comprises at least a part or all of amino acids 26-106 of SEQ ID NO. 1, e.g.26-100, 26-102, 26-104, 26-105 or 26-106, except for the mutations introduced as described. The F1 polypeptide comprises at least a part or all of amino acid residues 136 to 513 of SEQ ID NO. 1, for example 136 to 500, 140 to 500, 136 to 510, 136 to 512 or 136 to 513, except for the mutations introduced as described.
The F2 domain may be at position 100, position 101, position 102, position 103, position 104, position 105 or position 106 in the sequence of SEQ ID NO. 1. The C end of the F2 domain is directly connected with the N end of the F1 domain or is connected with the N end of the F1 domain through a Linker. The N-terminal of the F1 domain may be 136 th, 137 th, 138 th, 140 th, 141 th, 142 th, 143 th, 144 th, 145 th or 146 th bit of SEQ ID NO. 1. The C-terminus of the F1 domain may be position 499, 500, 502, 503, 504, 505, 506, 508, 510 or 513 of SEQ ID NO. 1. The C-terminal F1 domain can be linked to the trimerization domain or the multimerization domain directly or via a Linker.
The terms "multimerization domain" and "multimerization" refer to the formation of a dimer, trimer, tetramer, pentamer, hexamer, and/or tetracosane of a polypeptide or a domain of a polypeptide with other, more multimerization domains to form a polymer, trimerization domain such as foldon, GCN4, or MTQ.
In the present application, the term "GCN4" refers to the transcriptional activator GCN4 of yeast, a protein that binds to DNA through a leucine zipper (bZIP) structure, and the sequence of GCN4 in the present application is IKRMKQIEDKIEEIESKQKKIENEIARIKKIK.
In this application, the term "MTQ" is a trimerization module, in this application the sequence of MTQ is IKEEIAKIKEEQAKIKEKIAEIEKRIAEIEKRIAGGCC.
Example 1 plasmid construction, transformation and preparation
The sequence of the immunogenic RSV polypeptides, including wild-type (WT), ferritin, F ecto-Ferritin group (ectodomain of F protein+ferritin) and DS-Cav1B group were designed as shown in Table 1 below, and DS-Cav1A group was additionally designed by disulfide bond mutations S155C and S290C, and cavity filling mutations S190F and V207L on the basis of the WT sequence, and further P102A, I379V and M447V amino acid substitution mutations were performed on the sequence of DS-Cav1A group to obtain DS-Cav1B group.
The amino acid sequence was subjected to codon optimisation in accordance with the host CHO cell to determine the nucleic acid sequence for gene synthesis.
TABLE 1 amino acid sequence design of constructs
The synthesized target gene is inserted into eukaryotic plasmid pCHO3.1 vector for transfection and expression. Each recombinant plasmid constructed was transformed into E.coli DH 5. Alpha. Competent cells, glycerol bacteria transformed into plasmids containing the target gene were transferred into 150mL LB liquid medium (Amp final concentration 100. Mu.g/mL), shake cultured overnight at 37℃and 2000rpm, plasmid extraction was performed using endotoxin-free plasmid extraction kit, the plasmid concentration of the extract was measured, and enzyme digestion was performed, and specific reaction systems are shown in Table 2.
TABLE 2 cleavage reaction System
The concentration of the amplified plasmids is measured, and the measured concentration of each plasmid can obtain higher amplification concentration of about 600-900 mug/ml, and the total amount is about 1.0 mug. The cleavage results were detected by 1% agarose gel electrophoresis, and clear bands were visible at the corresponding size positions. All plasmids were sequenced and, as identified, all plasmids were completely correct in the gene sequence of interest. Recombinant plasmid vectors carrying the gene of interest have been successfully transferred into host cells and amplified.
The amplified concentration of the plasmid having intermolecular disulfide bond mutation was examined after the plasmid amplification, and the results are shown in the following table.
TABLE 3 plasmid concentration measurement results
As a result, the plasmid concentrations were relatively high, namely pCHO3.1-F46, pCHO3.1-F47, pCHO3.1-F48, pCHO3.1-F49 and pCHO3.1-F50.
Example 2 protein expression
The resulting plasmid was introduced into CHO cells by transient transfection, and protein supernatant was collected after culture.
ExpiCHO-S cells (Thermo) were cultured in EmCD CHO-S203 (Eminance/L20301) medium supplemented with 6mM L-glutamine (sigma/G5146), 96 well plates (supplemented with 10% FBS) were plated at 0.25E5 cells/well density one day in advance, 37℃at 8% CO 2 Is subjected to stationary culture in an incubator. The day of transfection, 96-well plates plated with cells were replaced with fresh L-glutamine-containing EmCD CHO-S203 medium. Preparing a transfection incubation system (volume is 50% of transfection volume) with EmCD CHO-S203 medium containing L-glutamine, wherein the amount of DNA is 1 μg/ml, the ratio of transfection reagent PEI (Polysciences/24765-1) to DNA is 3, mixing PEI and DNA, standing for 5min, incubating in 96-well plate with cells, culturing at 37deg.C and 8% CO 2 Is subjected to stationary culture in an incubator. After 24h of transfection, the temperature was reduced to 32℃for incubation, and a 5% by volume Feed supplement of Advanced Feed1 (sigma/24368-1L) was added. Culture was stopped at day 7 post-transfection to harvest supernatant.
Example 3 evaluation experiments on increased protein expression
And (3) carrying out molecular sieve chromatography by taking a DS-Cav1A protein supernatant sample stored at 4 ℃ and a DS-Cav1B protein supernatant sample as two groups of parallel samples to be tested. The height-diameter ratio of the molecular sieve chromatographic column is 40:1, and the filler is Superdex200; the mobile phase is 2.5% mercaptoethanol, 20mmol/L Tris-HCl with pH of 8.0; the flow rate is 50cm/hr; the sample loading amount is less than 5% (v/v) of the bed volume, and the flow-out peaks are collected step by step (chromatographic patterns are shown in figures 1 (A) and 1 (B), and electrophoresis results are shown in figures 2 (A) and 2 (B)). The applicant has unexpectedly found that, in contrast, the three natural substitutions of P102A, I379V and M447V of DS-Cav1B can greatly increase the expression level of the protein. DS-Cav1 used in the control experiments of the following examples refers to DS-Cav1B that is optimally expressed.
EXAMPLE 4F protein Individual mutagenesis protocol design
Each mutant of the present invention was designed and prepared based on the amino acid sequence of the wild-type (WT) RSV F protein described in SEQ ID NO. 1. This example illustrates the design of various F protein single mutants provided in the form of trimers, including foldon domains and introduced amino acid mutations, such as addition of non-natural disulfide mutations and cavity filling mutations. These F protein single mutants contained an IgG signal peptide (MGWSCIILFLVATATGVHS), two separate polypeptide chains. One of the polypeptide chains, the F2 polypeptide, comprises amino acids 26-106 of SEQ ID NO. 1, except for the mutations introduced as described. Another polypeptide chain comprises the F1 polypeptide (residues 136-513) linked to the N-terminus of the foldon domain, and their commonly introduced modifications include the substitution of del_P27 with ARGSG, R106Q and F137S in addition to the introduced mutations as described. Further included are purification tags His-Tag (HHHHHH), affinity Tag Monoclone Flag-Tag (DYKDDDK) and linker sequences (e.g., GSGSG). The signal peptide of SEQ ID NO. 1 (residues 1-25) and pep27 (residues 110-136) are cleaved from the F0 precursor during the expression process.
The amino acid sequences of the individual mutants of the RSV F protein and the control proteins were designed as follows, and the amino acid sequences were determined by codon optimization in accordance with the host CHO cells and were subjected to gene synthesis.
TABLE 4 mutant containing only added unnatural disulfide bonds
TABLE 5 mutants containing only cavity filling mutations
In addition, F protein single mutant only containing the linker sequence shown in the following table is designed, the C end of F2 domain and the N end of F1 domain are connected through the linker sequence, and the amino acid sequence is subjected to codon optimization according to the host CHO cell to determine the nucleic acid sequence and perform gene synthesis.
Template amino acid sequence:
MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIATVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK。
TABLE 6 design of linker sequences
Plasmid transformation, preparation and enzyme digestion identification were performed according to the method of example 1, and the concentration was measured after plasmid amplification, so that each plasmid could obtain a higher amplification concentration. All plasmids were sequenced and the sequence of the gene of interest was identified to be completely correct. Recombinant plasmid vectors carrying the gene of interest have been successfully transferred into host cells and amplified.
EXAMPLE 5 screening of intermolecular disulfide mutation schemes
The resulting plasmid was introduced into CHO cells by transient transfection, and protein supernatant was collected after culture. The coated antibody D25 or coated antibody motavizumab (abbreviated as mota) was diluted with PBS to 5 ng/. Mu.L, 200. Mu.L was added to each well of a 96-well plate, and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to an adaptation dilution (D25-1:8000, mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read.
TABLE 7 binding of mutants of different intermolecular disulfide mutation schemes to D25 and Mota
Selection of intermolecular disulfide bond designs preferably refers to the D25 antibody OD values, and the preferred proteins are F49, F54, F56, while referring to the mota antibody OD values, F50, F51, F54, F56, F49 are also preferred, and further binding to the amplified concentration measurements of plasmids having intermolecular disulfide bond mutations in the plasmid construction, preferred mutation designs include F49, F54, F50, and F56, i.e., preferred K75C + E218C, S C + Y458C, V144C + I407C and D486C + D489C intermolecular disulfide bond designs.
EXAMPLE 6 screening of RR1 disulfide mutation protocols
The following single mutant plasmids were introduced into CHO cells by transient transfection, and protein supernatants were collected after culture and allowed to stand at 4 ℃ for 7 days for detection.
The coated antibody D25 or coated antibody motavizumab (abbreviated as mota) was diluted with PBS to 5 ng/. Mu.L, 200. Mu.L was added to each well of a 96-well plate, and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to an adaptation dilution (D25-1:8000, mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read, and the sample concentration was calculated according to the regression analysis equation. The PreF conformational ratio of the protein after 7 days of standing at 4 ℃ was calculated and expressed as the ratio of D25 to Mota active concentration.
TABLE 8 PreF conformational duty cycle of proteins with RR1 disulfide mutations after 7 days of standing
The results show that F27, F25, F26 and F30 protein supernatants were able to detect higher PreF conformational ratios after 7 days at 4 ℃, indicating that they have an RR1 disulfide mutation scheme that is more favorable for maintaining the F protein pre-fusion conformation in a general vaccine storage environment, and therefore the preferred RR1 disulfide mutation schemes are v151c+i288C, H c+i291C, I148 7c+y286C and s55c+v188C.
EXAMPLE 7 screening of RR2 disulfide mutation protocols
The following single mutant plasmids were introduced into CHO cells by transient transfection, and protein supernatants were collected for detection after culturing.
The coated antibody, motavizumab (abbreviated as mota), was diluted with PBS to 5 ng/. Mu.L, and 200. Mu.L was added to each well of a 96-well plate and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to adaptation dilution (mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read.
TABLE 9 ability of proteins with RR2 disulfide mutations to bind Mota antibodies
The results show that F39, F42, F44, F41 and F45 are more capable of binding to Mota antibodies (one site II specific antibody that binds to both pre-and post-fusion F) compared to F39, F42, F44 and F45, and thus preferred RR2 disulfide mutation schemes are l321c+i475C, I332 336c+s485C, F c+i492C, I c+p480C and k399c + s485C.
EXAMPLE 8 design and screening of DS-Cav1 group RSV F mutant
The RSV F mutant proteins were designed according to the following table, based on DS-Cav1, in combination with cavity filling mutations, disulfide mutations. The s55c+l380c mutation is representative of an RR1 disulfide mutation, k399cjs485C and f387c+i492C are representative of an RR2 disulfide mutation, and v144c+i407C is representative of an intermolecular disulfide mutation.
TABLE 10 design of DS-Cav1 group RSV F mutant
The resulting plasmid was introduced into CHO cells by transient transfection, and protein supernatant was collected after culture. The protein supernatant concentration was diluted to the detection concentration and the pre-fusion conformational ratio of the protein was identified using specific mab D25 and Mota incubated at room temperature 25 ℃ and 50 ℃ for 1h.
The coated antibody D25 or the coated antibody motavizumab (abbreviated as mota) was diluted to 5 ng/. Mu.L with PBS, 200. Mu.L was added to each well and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to an adaptation dilution (D25-1:8000, mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST.
100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read, and the sample concentration was calculated according to the regression analysis equation.
TABLE 11 OD values and specific antibody Activity concentration for DS-Cav1 group mutant design
TABLE 12 OD values and specific antibody Activity concentration for DS-Cav1 group mutant design
The result shows that the RSV F mutant designed by further combining the exemplary cavity filling mutation or disulfide bond mutation on the basis of DS-Cav1 can be combined with the specific antibodies D25 and Mota both at room temperature and after incubation for 1h at 50 ℃, and basically has higher expression level compared with DS-Cav 1. Although the conformations are lost to varying degrees after heat treatment, they are relatively stable and maintain a high pre-fusion conformational ratio.
Example 9 combinatorial screening of intermolecular disulfide mutations evaluation of conformational thermal stability before F protein fusion
With reference to the design scheme of intermolecular disulfide bonds, further designing polypeptides introducing combinatorial mutations in RSV F ecto (ecto), the combinatorial screening of intermolecular disulfide bond mutations is focused on.
Preparing a large amount of expressed candidate protein, purifying by using a nickel column affinity chromatography and molecular sieve chromatography two-step purification method, diluting the protein concentration to a detection concentration, and identifying the Pre-fusion conformational stability of the purified protein by using Pre-F Pre-fusion conformational specificity monoclonal antibody D25 at 60 ℃ and 70 ℃ for 1 h.
Design of individual mutations of binding proteins the effect of the intermolecular disulfide bond (inter disulfide bond) mutation V144C + I407C on the conformational thermal stability of the F protein prior to fusion was first examined further. Conformational heat stability reference values were characterized using the sum of the D25 concentration ratios of different batches of protein after one hour of heat treatment to 4 ℃. The conformational thermal stability reference values in the following table are mainly the sum of the two batches, three times D25 concentration ratios, including the 0512 batch of protein at 60 ℃ for one hour and the 4 ℃ D25 concentration ratio at 0512 batch at 70 ℃ for one hour and the 4 ℃ D25 concentration ratio at 0509 batch at 70 ℃ for one hour, the results are shown in the table below.
TABLE 13 comparison of different disulfide bond designs and conformational thermal stability reference values
In terms of PreF conformational thermostability, the pre-fusion conformation of the V144C + I407C mutant design with other combinations of F proteins is relatively most stable in the design with inter disulfide compared to the F protein without the intermolecular disulfide design, followed by the F protein with the S150C + Y458C mutant design, which is incubated at 60 ℃ or 70 ℃ for 1 hour.
EXAMPLE 10 screening of combined disulfide mutations of RR1 bond and RR2 bond
Based on the preferred inter disulfide V144C + I407C further examining different combinations of different RR1 disulfide mutations and RR2 disulfide mutations, combined disulfide mutations were screened that could contribute to more pre-fusion conformations.
Preparing a large amount of expressed candidate proteins, purifying by using a nickel column affinity chromatography and molecular sieve chromatography two-step purification method, diluting the protein concentration to a detection concentration, detecting and calculating the protein activity of each expressed protein by adopting a double-antibody sandwich ELISA method and using 101F as a coated antibody and D25 and motavizumab (referred to as Mota for short) as labeled antibody detection pairing. Wherein, the protein activity is expressed as the concentration of the detection activity of the epitope specific neutralizing antibody, and the PreF conformational ratio is expressed as the ratio of the activity concentration of D25 to Mota of 0D, and the results are shown in the following table.
TABLE 14 combinatorial screening and detection of different disulfide bonds
The results show that introducing the combination of the RR1 disulfide mutation, I148c+y286C, with the RR2 disulfide mutation, F387c+i492C, on the same inter disulfide mutation, V144c+i407C, is the highest detected combination and is able to contribute to more pre-fusion conformations. In addition, there were no statistical differences in the combination of the RR1 disulfide mutation, I148C+Y286C and S55C+L188C, with the RR2 disulfide mutation, F387C+I 492C.
Example 11 influence of temperature on protein Properties
The D25 duty cycle (70-1 h/4-0D) after treatment at 70 ℃ was examined by heat treatment under the optimal combination of inter disulfide mutation, RR1 disulfide mutation and RR2 disulfide mutation, while examining different cavity filling mutations and appropriate design to increase expression level. The combination of cavity filling mutations preferably includes a regimen of N67F, for example: n67F, N67f+v495L, n67f+s190v+v495L or n67f+s190v+v296f+v495L. The substitution design scheme with increased expression level is p102a+i379v+m447V.
TABLE 15 screening design and detection of different combination mutations
The invention further provides multi-dimensional combinatorial screening, and examines the contribution of the combination of the mutation for increasing the expression level and the cavity filling mutation to the maintenance of the conformation before the fusion of the RSV F protein, and the result shows that the thermal stability of the PreF conformation of the protein with the combined cavity filling mutation N67F+S190V+V495L is far better than that of the protein with the single cavity filling mutation N67F under the extremely high temperature condition of 70 ℃. It was also observed that S46G, G184N and P102A+I379V+M447V also greatly aid in increasing protein expression and thermostability.
EXAMPLE 12 immunoassays
Female BALB/C mice of 6-8 weeks of age are selected for immunization, 6 mice in each group are randomly grouped, and vaccine double-spot immunization is carried out on the mice by intramuscular injection on days 0 and 21, and 50 mu L of each of the left leg and the right leg, wherein the immunogen comprises candidate antigen F147; DS-Cav1, pXCS847 (SEQ ID NO: 17) and PreF-1345 (SEQ ID NO: 18) positive controls; PBS negative control group. The antigen dose was 12. Mu.g/mouse, and the AS01 adjuvant dose was 1/20HD per mouse.
The AS01 adjuvant is in the form of liposome, and comprises main components of MPL, QS-21, DOPC and cholesterol, wherein the 1HD AS01 adjuvant is 0.5ml, and the components comprise 50 mug of MPL, 50 mug of QS-21, 1mg of DOPC, 0.25mg of cholesterol, 4.835mg of sodium chloride, 0.15mg of anhydrous disodium hydrogen phosphate, 0.54mg of monopotassium phosphate and water for injection. Serum was collected on day 20 and day 35, respectively, and the collected serum was stored at-20 ℃ for subsequent detection of bound antibodies.
Detection of bound antibodies:
(1) Diluting a serum sample to be detected by using a sample diluent;
(2) The positive control serum is diluted 1:10000 times by using a sample diluent, and the negative control serum is diluted 1:200 times by using a sample diluent;
(3) Sample adding: serial diluted serum sample to be tested, negative control serum, positive control serum and blank control solution (sample diluent is taken as blank control) are added into a 100 mul/Kong Jiazhi pre-F ELISA plate, 1 hole is added into the serum sample to be tested, and 2 holes are added into the control solution; igG-UNLB standard yeast ELISA strip at 100 μl/well was added to the sample dilution;
(4) Incubation: the ELISA plate is placed at 37 ℃ for 30 minutes, and is washed by washing liquid for 5 times in an automatic plate washing machine;
(5) Adding enzyme: placing an enzyme-labeled secondary antibody (HRP coat Anti-Mouse IgG (H+L), ABclonal) in a 100 μl/Kong Jiazhi enzyme-labeled plate, incubating at 37deg.C for 30 min, and washing the plate with washing liquid for 5 times;
(6) Color development: mixing the color development A solution and the color development B solution (flying organisms) in equal volume, and incubating in a 100 μl/Kong Jiazhi ELISA plate at room temperature in a dark place for 15 minutes;
(7) And (3) terminating: stop the reaction by adding a stop solution at 50. Mu.l/well;
(8) Reading a plate: the absorbance at a wavelength of 450nm (reference wavelength of 620 nm) was measured by a microplate reader, and the results are shown in the following table.
TABLE 16 Pre-F specific IgG content (3 weeks serum)
Note that: "/" means not measured or not detected
TABLE 17 Pre-F specific IgG content (5 week serum)
The results show that the Pre-F specific IgG content, F147 preF IgG GMC (geometric mean concentration) was highest, higher than the positive controls DS-Cav1 (see GSK company, preF design), PXCS847 (see Humus company design), and PreF-1345 (see Mudner company design) at 3 weeks serum detection with each group of immunogen in combination with adjuvant.
After boosting, pre-F IgG GMC (geometric mean concentration) was detected in the 5-week serum of each group of mice to a varying extent.
From the above experiments, it can be seen that the antigen and vaccine composition provided by the invention can induce the generation of neutralizing antibodies with high titer against RSV virus, which also suggests that the vaccine will provide better protective effect in the event of virus invasion, and has important significance for the development of RSV vaccine candidates.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (18)

1. An immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising an RSV F protein recombinant ectodomain corresponding to amino acid residues 26-513 of the wild-type RSV F protein sequence set forth in SEQ ID No. 1, wherein the recombinant ectodomain comprises a pair of disulfide mutations V144C + I407C, S C + N460C or a149C + Y458C; two other pairs of disulfide bond mutations are also included, wherein one mutation site in one pair of disulfide bonds is at positions 137 to 216 of the F protein, and one mutation site in the other pair of disulfide bonds is at positions 460 to 500 of the F protein.
2. The polypeptide of claim 1, wherein the pair of disulfide mutations at positions 137 to 216 of the F protein at the one mutation site is I148c+y286C, S c+l188C, H159c+i291C or g151c+i288C.
3. The polypeptide of claim 1, wherein the another pair of disulfide mutations at positions 460 to 500 of the F protein at the one mutation site is k399c+s485C, S c+s466C, T323c+i475C, I c+p480C or F387c+i492C.
4. An immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising an RSV F protein recombinant ectodomain corresponding to amino acid residues 26-513 of a wild-type RSV F protein sequence set forth in SEQ ID No. 1, wherein the recombinant ectodomain comprises a combination of disulfide mutations:
A. a disulfide mutation selected from V144C + I407C, S146C + N460C or a149C + Y458C;
B. a pair of disulfide mutations in positions 137 to 216 of the F protein at one mutation site; and
C. one of the mutation sites is a pair of disulfide mutations at positions 460 to 500 of the F protein.
5. The polypeptide of claim 4, wherein the pair of disulfide mutations at positions 137 to 216 of the F protein at one mutation site is I148c+y286C, S c+l188C, H159c+i291C or g151c+i288C; wherein the pair of disulfide mutations at positions 460 to 500 of the F protein at the one mutation site refers to K399C+S485C, S C+S466C, T323C+I475C, I C+P480C or F387C+I492C.
6. The polypeptide of claim 5, wherein the recombinant ectodomain comprises one of the following disulfide mutation combination schemes:
A. V144C + I407C, I C + Y286C and F387C + I492C;
B. V144C + I407C, I C + Y286C, S C + L188C and F387C + I492C.
7. The polypeptide of any one of claims 1-6, wherein the recombinant ectodomain further comprises a cavity-filling mutation N67F or n67f+s190v+v296f+v495L.
8. The polypeptide of claim 7, wherein the recombinant ectodomain further comprises an expression-enhancing mutation p102a+i379v+m V, S46G or G184N.
9. The polypeptide of any one of claims 1-8, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising at least amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising at least amino acid residues 145-513 of the RSV F protein.
10. The polypeptide of claim 9, wherein the recombinant F2 domain is linked to the recombinant F1 domain directly or through a linker.
11. The polypeptide of claim 10, wherein the linkage of the recombinant F2 domain to the recombinant F1 domain by linker means that the C-terminal of the F2 domain and the N-terminal of the F1 domain are substituted by linker selected from GGPGGS, GAPEPGE or GGSGGSG.
12. The polypeptide of any one of claims 1-11, wherein the C-terminus of the polypeptide is linked to a trimerization domain or a multimerization domain.
13. The polypeptide of claim 12, wherein the trimerization domain is a foldon, GCN4, or MTQ domain.
14. The polypeptide of claim 12, wherein the multimerization domain is a ferritin domain.
15. Use of the polypeptide of any one of claims 1-14 in the preparation of a vaccine for preventing RSV infection.
16. A vaccine for preventing RSV infection comprising the polypeptide of any one of claims 1-14; the vaccine provides protection against infection by at least one of the RSV subtypes a and/or B.
17. The vaccine of claim 16, comprising an adjuvant in the form of liposomes that prevents RSV infection.
18. The vaccine of claim 17, wherein the components of the liposomal adjuvant comprise MPL, QS-21, and cholesterol.
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