Respiratory syncytial virus reconstructed polypeptide and its application

Abstract

The present invention relates to a respiratory syncytial virus (RSV) F protein reconstructed polypeptide, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell transformed or transfected with the above polynucleotide, nucleic acid construct or expression vector, a stabilized polymer formed by the reconstructed polypeptide, an immunogenic composition comprising any of the foregoing, and its use in preparing a vaccine for preventing and / or treating respiratory syncytial virus infection. The RSV F protein reconstructed polypeptide of the present invention has good immunogenicity, can stimulate the body to produce a high level of neutralizing antibody titer, and is of great significance for the clinical treatment and prevention and control of respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to a respiratory syncytial virus reconstructed polypeptide, a respiratory syncytial virus recombinant antigen formed thereby, an immunogenic composition containing the same, and a preparation method and use thereof. Background Art

[0002] Human respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infections in infants and premature infants worldwide. Currently, there is an increasing demand for a safe and effective RSV vaccine worldwide.

[0003] RSV fusion protein (F protein) is a key mediator of RSV virion fusion with host cells. It exhibits extremely high immunogenicity in the state before fusion of virion and host cells and is considered an ideal target for RSV vaccine development. It has been identified that the stable RSV F protein in the pre-fusion conformation produces a stronger neutralizing immune response than the RSV F protein in the post-fusion conformation.

[0004] There have been a lot of studies on the stability of RSV F protein before fusion, but most of them focus on mutating the amino acids at specific sites of RSV F protein or introducing a trimerization domain at the C-terminus of F protein. In previous studies, the method of introducing a trimerization domain at the C-terminus of F protein was to replace the transmembrane domain (TM) of F protein with the trimerization domain. However, after the introduction of the trimerization domain, the effect of the deletion of the transmembrane domain on the protective effect of the vaccine has not been studied in depth. Summary of the invention

[0005] The present invention provides a reconstructed polypeptide based on the prefusion F protein of respiratory syncytial virus (RSV), a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell transformed or transfected with the above-mentioned polynucleotide, nucleic acid construct or expression vector, a stabilized multimer formed by the reconstructed polypeptide, an immunogenic composition comprising any of the foregoing, and use thereof in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus infection.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a respiratory syncytial virus (RSV) reconstructed polypeptide, wherein the RSV F protein reconstructed polypeptide includes the following domains directly connected to each other or connected by a linker from the N-terminus to the C-terminus: an extracellular domain of the RSV F protein, a trimerization domain, and a transmembrane domain of the RSV F protein; wherein the trimerization domain insertion site is: the corresponding site of the amino acid 513 of the RSV F protein precursor polypeptide (F0), and the transmembrane domain is a homologous transmembrane domain or a heterologous transmembrane domain of the RSV F protein.

[0008] For the above RSV F protein reconstructed polypeptide, the RSV F protein can be a F0 protein derived from any RSV subtype or any RSV strain, for example, it can be a wild-type F protein of RSV A2 strain (Uniprot number: P03420), B1 strain (Uniprot number: O36634), 18537 strain (Uniprot number: P13843), TX-79233 strain (Uniprot number: R9TCY6), etc. Exemplary F0 protein sequences are shown in SEQ ID NOs: 1-4.

[0009] In addition, the RSV F protein can also be a mutant of the F0 protein; the mutant of the F protein can be any (PreF) mutant that maintains the pre-fusion conformation of the F protein, for example, it can be a PreF mutant recorded in the following literature: "McLellan, Jason S., et al. science 342.6158 ((2013)): 592-598", "Krarup, Anders, et al. Nature communications 6.1 ((2015)): 8143", "Joyce, M. Gordon, et al. Nature structural &molecular biology 23.9 ((2016)): 811-820", "Che, Ye, et al. Science Translational Medicine 15.693 ((2023)): eade6422". Preferably, the RSV F protein is a F protein mutant having at least 80% amino acid sequence identity with the corresponding wild-type F protein.

[0010] In a feasible embodiment, the RSV F protein is the F protein of RSV virus type A or type B, or a mutant thereof having at least 80% sequence identity thereto.

[0011] In a feasible embodiment, the trimerization domain is selected from:

[0012] Bacteriophage T4 minor fibrin foldon (T4 foldon) and variants thereof (e.g., its sequence is as shown in SEQ ID NO: 5), MTQ (e.g., its sequence is as shown in SEQ ID NO: 6), heptapeptide ideal three-chain coiled coil (e.g., its sequence is as shown in SEQ ID NO: 7), isoleucine zipper (IZ) long chain (e.g., its sequence is as shown in SEQ ID NO: 8), IZ short chain (e.g., its sequence is as shown in SEQ ID NO: 9), leucine zipper GCN4 and variants thereof (e.g., its sequence is as shown in SEQ ID NO: 10), pulmonary surfactant-associated protein D trimerization domain (e.g., its sequence is as shown in SEQ ID NO: 11), collagen trimerization domain (e.g., its sequence is as shown in SEQ ID NO: 12), cartilage matrix protein-1 long chain trimerization domain (e.g., its sequence is as shown in SEQ ID NO: 13) or cartilage matrix protein-1 short chain trimerization domain (e.g., its sequence is as shown in SEQ ID NO: 14) or other trimerization domains.

[0013] In a feasible embodiment, the transmembrane domain of the RSV F protein is a homologous transmembrane domain F / TM of the F protein (for example, its sequence is as shown in SEQ ID NO: 60, 72, 73), or a heterologous transmembrane domain, such as the transmembrane domain gp160 / TM of the HIV-1 virus gp160 protein (for example, as shown in SEQ ID NO: 61), the transmembrane domain HA / TM of the influenza virus HA protein (for example, its sequence is as shown in SEQ ID NO: 62), the transmembrane domain S / TM of the new coronavirus S protein (for example, its sequence is as shown in SEQ ID NO: 63), or other transmembrane domains.

[0014] In addition, in a feasible embodiment, the C-terminus of the transmembrane domain also comprises the intracellular domain of the protein from which it is derived. For example, when the transmembrane domain is a homologous transmembrane domain of the RSV F protein, the C-terminus of the homologous transmembrane domain also comprises the intracellular domain of the RSV F protein; when the transmembrane domain is a heterologous transmembrane domain, the C-terminus of the heterologous transmembrane domain also comprises the intracellular domain of the heterologous protein from which it is derived.

[0015] Furthermore, in a feasible embodiment, the linker has an amino acid sequence as shown in SEQ ID NO: 64 or SEQ ID NO: 65.

[0016] In a preferred embodiment, the RSV F protein reconstructed polypeptide has an amino acid sequence selected from the group consisting of:

[0017] SEQ ID NO: 24, 27, 30, 33, 35, 37, 38, 39, 40, 74.

[0018] In a feasible embodiment, the RSV F protein reconstructed polypeptide further comprises a signal peptide, which may be a homologous signal peptide or a heterologous signal peptide; optionally, the heterologous signal peptide is an EV preM signal peptide, a VSV-G signal peptide, an IgE heavy chain signal peptide, an IgK light chain signal peptide, a Secrecon signal peptide, a CD33 signal peptide, or a tissue-type plasminogen activator tPA signal peptide.

[0019] Preferably, the homologous signal peptide has an amino acid sequence as shown in SEQ ID NO: 66 or 67;

[0020] Preferably, the heterologous signal peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 15-21;

[0021] Preferably, the signal peptide is located at the N-terminus.

[0022] In a second aspect, the present invention provides a polynucleotide encoding the RSV F protein reconstructed polypeptide as described in the first aspect above.

[0023] In a specific embodiment, the polynucleotide may be DNA or mRNA; preferably, the polynucleotide is a nucleotide sequence optimized for human codons.

[0024] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and at least one expression control element operably linked to the polynucleotide.

[0025] In some embodiments, the nucleic acid construct comprises or consists of a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75, or an RNA sequence corresponding to these sequences.

[0026] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0027] In a fifth aspect, the present invention provides a host cell, wherein the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect is transformed or transfected;

[0028] Optionally, the host cell is a mammalian cell, an insect cell, a yeast cell or a bacterial cell;

[0029] Further optionally, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell;

[0030] Further optionally, the bacterial cell is an Escherichia coli cell.

[0031] In a sixth aspect, the present invention provides a respiratory syncytial virus (RSV) recombinant antigen, wherein the RSV recombinant antigen comprises a multimer, preferably a trimer, of the RSV F protein reconstructed polypeptide as described in the first aspect above.

[0032] In the seventh aspect, the present invention provides the use of the RSV F protein reconstructed polypeptide as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the RSV recombinant antigen as described in the sixth aspect in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus infection.

[0033] In a feasible embodiment, in the vaccine, the RSV F protein reconstructed polypeptide as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, or the RSV recombinant antigen as described in the sixth aspect above is the only antigenic component.

[0034] In other feasible embodiments, in addition to the RSV F protein reconstructed polypeptide as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, or the RSV recombinant antigen as described in the sixth aspect above, the vaccine further comprises other antigen components.

[0035] In an eighth aspect, the present invention provides a vaccine or immunogenic composition, comprising the RSV F protein reconstructed polypeptide as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, or the RSV recombinant antigen as described in the sixth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0036] In some preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus recombinant protein vaccine, which includes the RSV F protein reconstructed polypeptide as described in the first aspect above or the RSV recombinant antigen as described in the sixth aspect above, and an adjuvant;

[0037] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, Matrix M2 adjuvant, GLA-SE adjuvant.

[0038] In other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus DNA vaccine, which comprises:

[0039] (i) a eukaryotic expression vector comprising a DNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above; and

[0040] (ii) DNA vaccine adjuvants;

[0041] Optionally, the DNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above is a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

[0042] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors;

[0043] Optionally, the DNA vaccine adjuvant is selected from: human granulocyte macrophage colony stimulating factor, human interleukin-12 and / or CpG oligonucleotide.

[0044] In other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus mRNA vaccine, and the mRNA vaccine comprises:

[0045] (I) an mRNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above;

[0046] (II) mRNA vaccine adjuvants; and

[0047] (III) a delivery carrier, wherein the delivery carrier is selected from one or a combination of lipid nanoparticles, cationic nanoemulsions, polypeptides, polymers, cationic polypeptide polymers, and cationic polypeptide lipid nanoparticles.

[0048] Optionally, the mRNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above is an mRNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

[0049] Optionally, the mRNA vaccine adjuvant is selected from: lipid nanoparticles, emulsions, Toll-like receptor agonists and / or CpG oligonucleotides.

[0050] In other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus-viral vector vaccine, which comprises:

[0051] (1) viral backbone vectors; and

[0052] (2) a DNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above, which is constructed into the viral backbone vector;

[0053] Optionally, the DNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above is a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

[0054] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

[0055] In other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus nanoparticle vaccine, which comprises the RSV F protein reconstructed polypeptide and a nanoparticle carrier as described in the first aspect above.

[0056] Optionally, the nanoparticle carrier is ferritin, and the RSV F protein reconstructed polypeptide described in the first aspect is covalently linked to ferritin and self-assembled into nanoparticles, so that the RSV F protein reconstructed polypeptide is presented on the surface of the nanoparticles.

[0057] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, an oral preparation or a parenteral preparation;

[0058] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0059] Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, soft / hard capsules, film coatings and ointments;

[0060] Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or a pushable preparation.

[0061] In a ninth aspect, the present invention provides a method for preparing the RSV F protein reconstructed polypeptide as described in the first aspect above, characterized in that the preparation method comprises:

[0062] A nucleotide sequence encoding a signal peptide is added to the 5' end of the codon-optimized nucleotide sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above, and a nucleotide sequence encoding a histidine tag and a stop codon are added to the 3' end, and cloned and expressed, the correct recombinant is screened, and then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate and obtain the RSV F protein reconstructed polypeptide.

[0063] In a possible implementation of the above method, the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells; optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells; optionally, the bacterial cells are Escherichia coli cells.

[0064] In the tenth aspect, the present invention provides a method for preventing and / or treating respiratory syncytial virus infection, the method comprising: administering to a subject in need thereof a preventive and / or therapeutically effective amount of the following substances: the RSV F protein reconstructed polypeptide as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, the RSV recombinant antigen as described in the sixth aspect above, and / or the vaccine or immunogenic composition as described in the eighth aspect above.

[0065] The "preventively and / or therapeutically effective amount" may vary depending on the subject of administration, the subject's organ, symptoms, administration method, etc., and can be determined based on the veterinarian's judgment, taking into account the type of dosage form, administration method, age and weight of the subject, symptoms of the subject, etc.

[0066] Beneficial Effects

[0067] The present invention introduces a trimerization domain at a specific site of the RSV pre-fusion F protein, and connects the homologous transmembrane domain or heterologous transmembrane domain of the RSV F protein at the C-terminus of the trimerization domain, and designs the RSV F protein reconstruction polypeptide of the present invention; Experiments have shown that the pre-fusion conformation of the RSV F protein reconstruction polypeptide of the present invention is more stable, and the stability of the trimerization protein formed by it is also enhanced, thereby resulting in stronger immunogenicity and higher immune response effect. The RSV F protein reconstruction polypeptide of the present invention has good immunogenicity, can stimulate the body to produce a high level of neutralizing antibody titer, and is of great significance for the clinical treatment and prevention and control of respiratory syncytial virus.

[0068] In addition, the present invention experimentally confirmed that the combination of different trimerization domains and transmembrane domains from different sources can induce a higher level of neutralizing antibody titer, which shows that the above design method does not depend on a specific RSV subtype or a specific F protein or its mutant, and can achieve a higher level of neutralizing antibody titer in a variety of different mutants. In short, the RSV F protein reconstructed polypeptide of the present invention has a broad spectrum of effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0070] Figure 1 A schematic diagram showing the structure of the polypeptide described in Example 1.

[0071] Figure 2 A schematic diagram of the mRNA structure described in Example 2 is shown.

[0072] Figure 3 Representative ELISA results of the expression of mRNA-LNP encoding the prefusion F protein (PreF) mutant described in Example 3 in HEK293 cells are shown.

[0073] Figure 4 Representative flow cytometry results of mRNA-LNP encoding the prefusion F protein (PreF) mutant described in Example 3 expressed in HEK293 cells and labeled with PreFø epitope antibody are shown.

[0074] Figure 5 Representative flow cytometry results of mRNA-LNP encoding the prefusion F protein (PreF) mutant described in Example 3 expressed in HEK293 cells and labeled with an F protein IV epitope antibody are shown.

[0075] Figure 6 The immunogenicity of the mRNA-LNPs encoding different PreF protein mutants containing the trimerization domain of the bacteriophage T4 minor fibrin foldon (T4 foldon) recorded in Example 5 in mice is shown; wherein, AD respectively show the neutralizing antibody titers of the sera of mice immunized with mRNA-LNPs encoding different PreF mutants PreF1, PreF2, PreF3, and PreF4; the neutralizing antibody titers are displayed as NT50 geometric mean titer (GMT) bar graphs, and the GMT values ​​are marked on the top of the bar graphs, and the error bars represent 95% CI (confidence intervals).

[0076] Figure 7 The immunogenicity of mRNA-LNPs encoding PreF protein mutants containing MTQ or isoleucine zipper (IZ) short chain trimerization domains described in Example 5 in mice is shown; the figure shows mRNA-LNPs encoding PreF1-IZ, PreF1-IZ-F / TM A 、PreF1-MTQ、PreF1-MTQ-F / TM ANeutralizing antibody titers of sera from mice immunized with mRNA-LNP. Neutralizing antibody titers are shown as NT50 GMT bar graphs, with GMT values ​​annotated at the top of the bar graphs and error bars representing 95% CI.

[0077] Figure 8 The immunogenicity of mRNA-LNPs encoding PreF mutants containing different transmembrane domains (TM) described in Example 5 in mice is shown; the figure shows PreF protein mutants PreF1-T4-HA / TM, PreF1-T4-S / TM, PreF1-T4-gp160 / TM, PreF1-T4-F / TM encoding TM containing influenza HA or novel coronavirus S protein, HIV-1 gp160, and RSV F protein A 、PreF1-T4-F / TM A525-550 As well as PreF1-T4 and PreF1-F / TM as controls A Neutralizing antibody titers of sera from mice immunized with mRNA-LNP. Neutralizing antibody titers are shown as NT50 GMT bar graphs, with GMT values ​​annotated at the top of the bar graphs and error bars representing 95% CI. DETAILED DESCRIPTION

[0078] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0079] The term "polypeptide" refers to any amino acid chain, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers, including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as those in which one or more amino acid residues are non-natural amino acids, e.g., artificial chemical mimetics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimetic that is incorporated into a polypeptide via an amide bond or an amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxyl terminal (C-terminal) end. "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0080] The term "F protein" or "F polypeptide" refers to a protein or polypeptide having all or part of the amino acid sequence of RSV F protein.

[0081] RSV F protein, also known as Fusion protein or fusion protein, is an RSV envelope glycoprotein that can promote the fusion of RSV viral membrane and host cell membrane, and plays a vital role in the respiratory syncytial virus infection process. In nature, RSV F protein is initially synthesized as a single polypeptide precursor of approximately 574 amino acids in length, named F0. F0 includes an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the signal peptide (approximately the first 25 residues of F0) is proteolytically cleaved. The remaining F0 residues form trimers by oligomerization and are synthesized at two conserved consensus furin cleavage sequences (approximately located at positions 109 and 136 of F0; for example, RARR 109 (amino acid residues 106-109 of F0) and RKRR 136 (amino acid residues 133-136 of F0) are proteolytically processed by cellular proteases to produce two disulfide-linked fragments F1 and F2. The smaller of these fragments, F2, is derived from the N-terminal portion of the F0 precursor and includes approximately amino acid residues 26-109 of F0. The larger of these fragments, F1, includes the C-terminal portion of the F0 precursor (approximately amino acid residues 137-574 of F0), including the extracellular / luminal region (approximately amino acid residues 137-529), the transmembrane domain (approximately amino acid residues 525-550), and the C-terminal cytoplasmic tail (approximately amino acid residues 551-574). The extracellular portion of the RSV F protein is the RSV F ectodomain, which includes the F2 protein and the F1 ectodomain.

[0082] RSV F protein shows significant sequence conservation in RSV subtypes. For example, in F0 precursor molecules, RSV subtypes A and B have 90% sequence identity, and RSV subtypes A and B each have 81% sequence identity with cattle RSV F protein. In RSV subtypes, F0 sequence identity is even higher; For example, in each subtype in RSV A, B and cattle subtypes, RSV F0 precursor protein has about 98% sequence identity. Almost all identified RSV F0 precursor protein lengths are about 574 amino acids, and the slight differences in length are generally due to the length of the C-terminal cytoplasmic tail. In view of the conservation of RSV F sequences, those of ordinary skill in the art can easily compare the amino acid positions between different natural RSV F sequences to identify the corresponding RSV F amino acid positions between different RSV strains and subtypes. Therefore, the conservation of the RSV F protein sequence across strains and subtypes allows the use of reference RSV F sequences to compare the amino acids at specific positions in RSV F protein. Unless the context indicates otherwise, the numbering of the amino acid substitutions disclosed herein is by reference to the RSV F0 polypeptide sequence set forth in SEQ ID NOs: 1-4.

[0083] Three F2-F1 protomers oligomerize in the mature F protein, which adopts a metastable prefusion conformation that is triggered to undergo a conformational change to a postfusion conformation upon contact with the target cell membrane. This conformational change exposes a hydrophobic sequence, called the fusion peptide, which is located at the N-terminus of the F1 extracellular domain and binds to the host cell membrane and promotes the fusion of the membrane of the virus or infected cell with the target cell membrane.

[0084] A "domain" or "domain" of a polypeptide or protein refers to a structurally defined element within a polypeptide or protein, for example, a "trimerization domain" refers to an amino acid sequence within a polypeptide that promotes the assembly of a polypeptide into a trimer. For example, a trimerization domain can promote assembly into a trimer via association with other trimerization domains (of other polypeptides having the same or different amino acid sequences); a "transmembrane domain" refers to an amino acid sequence that can be inserted into a lipid bilayer (e.g., a lipid bilayer of a cell or virus or virus-like particle) or anchor an antigen to a membrane. The term is also used to refer to a polynucleotide encoding such a peptide or polypeptide. The above-mentioned domains in the RSV F protein reconstructed polypeptide of the present invention can be directly connected, or can be connected by a natural or artificial linker, linker, or spacer.

[0085] The term "linker" or "joint" or "spacer" or "interval sequence" is a natural or artificial bifunctional molecule that can be used to connect two molecules into one continuous molecule. Non-limiting examples of peptide linkers include glycine-serine peptide linkers and fragments of any length between amino acids 514-524 of RSV F protein. Unless otherwise indicated in the context, reference to "connecting" a first polypeptide to a second polypeptide, or "connecting" two polypeptides together, or a first polypeptide "connected" to a second polypeptide, refers to a covalent connection through a peptide bond (e.g., through a peptide linker) so that the first and second polypeptides form a continuous polypeptide chain. If a peptide linker is involved, the covalent connection of the first and second polypeptides can be connected to the N-terminus and C-terminus of the peptide linker. The term "mutant" refers to a molecule whose amino acid sequence is different from a natural or reference sequence. Amino acid sequence variants may have substitutions, deletions and / or insertions at specific positions within the amino acid sequence compared to a natural or reference sequence. Typically, variants have at least 50% homology with a natural or reference sequence. In some embodiments, variants have at least 80% or at least 90% homology with a natural or reference sequence.

[0086] The term "homology" refers to the overall correlation between polymeric molecules, such as nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymeric molecules (such as nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules) that have a critical level of similarity or identity determined by the comparison of matching residues are referred to as homology. Homology is a qualitative term describing the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term defining the degree of sequence matching between two comparison sequences. In some embodiments, if the sequence of a polymeric molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% consistent or similar, it is considered to be "homologous" to each other. The term "homology" must refer to the comparison between at least two sequences (polynucleotide or polypeptide sequences). If the polypeptides encoded by the two polynucleotide sequences are at least 50%, 60%, 70%, 80%, 90%, 95% or even 99% identical for at least one stretch of at least 20 amino acids, the two sequences are considered to be homologous. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode stretches with at least 4 to 5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode stretches with at least 4 to 5 uniquely specified amino acids. If the two proteins are at least 50%, 60%, 70%, 80% or 90% identical for at least one stretch of at least 20 amino acids, the two protein sequences are considered to be homologous. The terms "polynucleotide" and "nucleic acid sequence" refer to a nucleotide polymeric form with a length of at least 10 bases. Nucleotides can be ribonucleotides, deoxyribonucleotides, or a modified form of any nucleotide. The 5' and 3' directions of nucleic acids are defined by reference to the connectivity of each nucleotide unit and are assigned according to the carbon position of the deoxyribose (or ribose) sugar ring. The information (coding) content of a polynucleotide sequence is read in the 5' to 3' direction.

[0087] The term "reconstructed polypeptide" or "recombinant polypeptide" refers to a polypeptide having a non-naturally occurring sequence or a sequence generated by artificially combining two or more otherwise separate sequence segments, which may be in the same protein or in different proteins.

[0088] In some embodiments, the nucleotide vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one respiratory syncytial virus (RSV) antigenic polypeptide, wherein the RNA comprises at least one chemical modification.

[0089] The terms "chemical modification" and "chemically modified" refer to modifications of at least one of the positions, patterns, percentages or populations of adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides. Typically, these terms do not refer to ribonucleotide modifications of the naturally occurring 5' terminal mRNA cap portion. Modifications of polynucleotides include, but are not limited to, those described herein, and include (but are not explicitly limited to) those modifications comprising chemical modifications. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprise naturally occurring, non-naturally occurring modifications or polynucleotides may comprise a combination of naturally occurring modifications and non-naturally occurring modifications. Polynucleotides may include any applicable modifications to, for example, sugars, nucleobases or internucleoside linkages (e.g., to connecting phosphates, to phosphodiester linkages or to phosphodiester backbones).

[0090] The term "heterologous" with respect to nucleic acids, polypeptides or other cellular components indicates the presence of components that are not normally found in nature and / or that originate from a different source or species.

[0091] "Antigen" refers to a compound, composition, or substance that can stimulate antibody production and / or T cell response in an animal, including a composition introduced into an animal by injection, adsorption, or other means. The term "antigen" includes all relevant antigenic epitopes. The term "epitope" or "antigenic determinant" refers to a site on an antigen that is responded to by B and / or T cells. "Predominant antigenic epitope" or "predominant epitope" refers to those epitopes to which a functionally significant host immune response (e.g., antibody response or T cell response) is produced. Thus, with respect to a protective immune response against a pathogen, a dominant antigenic epitope refers to those antigenic modules that produce protection from diseases caused by the pathogen when recognized by the host immune system. The term "T cell epitope" refers to an epitope that is specifically bound by a T cell (via a T cell receptor) when bound to a suitable MHC molecule. "B cell epitope" refers to an epitope that is specifically bound by an antibody (or B cell receptor molecule).

[0092] "Vaccine" refers to an immunogenic material preparation capable of stimulating an immune response, which is administered for the prevention, improvement or treatment of infectious diseases or other types of diseases. Immunogenic substances may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived therefrom. Vaccines may include disclosed immunogens (such as recombinant RSVF extracellular domain trimers or nucleic acid molecules encoding them), viruses, cells or one or more cell components. Vaccines may induce preventive (preventive or protective) and therapeutic responses. The method of administration varies according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. The vaccine may be administered together with an adjuvant to enhance the immune response. In a specific non-limiting example, the vaccine prevents and / or reduces the severity of symptoms associated with RSV infection and / or reduces viral load compared to a control.

[0093] "Carrier" or "excipient" refers to pharmaceutically acceptable excipients, which refer to excipients and additives used in the production of drugs and the preparation of prescriptions; in addition to the active ingredients, they are substances that have been reasonably evaluated in terms of safety and are included in drug preparations. In addition to excipients, acting as carriers, and improving stability, "pharmaceutically acceptable excipients" also have important functions such as solubilization, dissolution assistance, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their functions and uses, pharmaceutical excipients can be divided into solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0094] "Recombinant" is a molecule with a sequence that does not occur naturally, for example, including one or more nucleic acid substitutions, deletions or insertions, and / or with a sequence that is prepared by the artificial combination of two otherwise isolated sequence segments. This artificial combination can be accomplished by chemical synthesis or more commonly by artificial manipulation of isolated nucleic acid segments (e.g., by genetic engineering techniques). A recombinant protein is a protein with a sequence that does not occur naturally or with a sequence that is prepared by the artificial combination of two otherwise isolated sequence segments. In several embodiments, the recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell (e.g., a bacterial or eukaryotic cell), or into the genome of a recombinant virus.

[0095] "Expression control element" refers to a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operably connected thereto. When the expression control sequence controls and regulates the transcription and (appropriate) translation of the nucleic acid sequence, the expression control sequence is operably connected to the nucleic acid sequence. Therefore, the expression control sequence may include a suitable promoter, enhancer, transcription terminator, a start codon (ATG) before a protein coding gene, a splicing signal of an intron (maintaining the correct reading frame of the gene to allow correct translation of mRNA) and a stop codon. The term "regulatory element" is intended to include at least components whose presence may affect expression, and may also include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence. The expression control element may include a promoter.

[0096] "Promoter" is a minimal sequence sufficient to direct transcription. It includes T7 promoters, and also includes promoter elements that are sufficient to make promoter-dependent gene expression controllable with respect to cell type specificity, tissue specificity, or inducible by external signals or agents; such elements can be located at the 5' or 3' of the gene. It includes constitutive and inducible promoters (see, for example, Bitter et al., Methods in Enzymology 153: 516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of phage λ can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from mammalian cell genomes (such as metallothionein promoters) or promoters derived from mammalian viruses (such as retroviral long terminal repeats; adenovirus late promoters; vaccinia virus 7.5K promoters) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of nucleic acid sequences.

[0097] "5'UTR" refers to the region of an mRNA that is located directly upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript translated by the ribosome) and does not encode a polypeptide. "3'UTR" refers to the region of an mRNA that is located directly downstream (i.e., 3') of the stop codon (i.e., the codon of an mRNA transcript that transmits a translation termination signal) and does not encode a polypeptide.

[0098] A "coding sequence CDS" is a continuous stretch of RNA that begins with a start codon, such as methionine (AUG), and ends with a stop codon, such as UAA, UAG, or UGA, and encodes a polypeptide.

[0099] A "polyA tail" or "poly A tail" is located in the downstream region of the 3'UTR in the mRNA and contains multiple consecutive adenosine monophosphates. The polyA tail can contain 10 to 300 adenosine monophosphates. For example, the polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, the polyA tail contains 50 to 250 adenosine monophosphates. In some embodiments, the non-adenosine nucleotides in the polyA tail segment the consecutive adenosine monophosphates. In relevant biological contexts (eg, in cells, in vivo), the poly(A) tail serves to protect the mRNA from enzymatic degradation, such as in the cytoplasm, and facilitates transcription termination and / or mRNA export from the nucleus and translation.

[0100] "Host cell" refers to a cell in which a vector can be propagated and express the vector nucleic acid. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It should be understood that all progeny may be different from the parent cell because mutations may occur during the replication process. However, when the term "host cell" is used, such progeny are included.

[0101] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0102] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0103] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0104] Example 1: Structural design of reconstructed polypeptides

[0105] In this example, the following reconstructed polypeptides of the present invention and control polypeptides thereof were designed respectively.

[0106] The reconstructed polypeptide of the present invention:

[0107] PreF1-T4-F / TM A (whose sequence is shown in SEQ ID NO: 24), PreF2-T4-F / TM A (whose sequence is shown in SEQ ID NO: 27), PreF3-T4-F / TM A (whose sequence is shown in SEQ ID NO: 30), PreF4-T4-F / TM B (whose sequence is shown in SEQ ID NO: 33), PreF1-MTQ-F / TM A (whose sequence is shown in SEQ ID NO: 35), PreF1-IZ-F / TM A (whose sequence is shown in SEQ ID NO: 37), PreF1-T4-gP160 / TM (whose sequence is shown in SEQ ID NO: 38), PreF1-T4-HA / TM (whose sequence is shown in SEQ ID NO: 39), PreF1-T4-S / TM (whose sequence is shown in SEQ ID NO: 40), PreF1-T4-F / TM A525-550 (Its sequence is shown in SEQ ID NO: 74).

[0108] Control peptide:

[0109] PreF1-F / TM A (whose sequence is shown in SEQ ID NO: 22), PreF1-T4 (whose sequence is shown in SEQ ID NO: 23), PreF2-F / TM A (whose sequence is shown in SEQ ID NO: 25), PreF2-T4 (whose sequence is shown in SEQ ID NO: 26), PreF3-F / TM A (whose sequence is shown in SEQ ID NO: 28), PreF3-T4 (whose sequence is shown in SEQ ID NO: 29), PreF4-F / TM B (whose sequence is shown in SEQ ID NO: 31), PreF4-T4 (whose sequence is shown in SEQ ID NO: 32), PreF1-MTQ (whose sequence is shown in SEQ ID NO: 34), and PreF1-IZ (whose sequence is shown in SEQ ID NO: 36).

[0110] The sequences of the above polypeptides are all sequences that do not contain their endogenous signal peptides, and the endogenous signal peptide sequences used by them are shown in SEQ ID NO: 66 or 67; wherein PreF1-F / TMA (whose sequence is shown in SEQ ID NO: 22), PreF1-T4 (whose sequence is shown in SEQ ID NO: 23), PreF1-T4-F / TM A (whose sequence is shown in SEQ ID NO: 24), PreF2-F / TM A (whose sequence is shown in SEQ ID NO: 25), PreF2-T4 (whose sequence is shown in SEQ ID NO: 26), PreF2-T4-F / TM A (whose sequence is shown in SEQ ID NO: 27), PreF3-F / TM A (whose sequence is shown in SEQ ID NO: 28), PreF3-T4 (whose sequence is shown in SEQ ID NO: 29), PreF3-T4-F / TM A (whose sequence is shown in SEQ ID NO: 30), PreF1-MTQ (whose sequence is shown in SEQ ID NO: 34), PreF1-MTQ-F / TM A (whose sequence is shown in SEQ ID NO: 35), PreF1-IZ (whose sequence is shown in SEQ ID NO: 36), PreF1-IZ-F / TM A (whose sequence is shown in SEQ ID NO: 37), PreF1-T4-gP160 / TM (whose sequence is shown in SEQ ID NO: 38), PreF1-T4-HA / TM (whose sequence is shown in SEQ ID NO: 39), PreF1-T4-S / TM (whose sequence is shown in SEQ ID NO: 40), PreF1-T4-F / TM A525-550 The endogenous signal peptide sequence of the polypeptide is shown in SEQ ID NO: 74. The endogenous signal peptide sequences of the remaining polypeptides are shown in SEQ ID NO: 67.

[0111] The structural diagrams of the above polypeptides are shown in Figure 1 shown.

[0112] Example 2: Preparation of mRNA for reconstructed polypeptide and packaging of lipid nanoparticles (LNP)

[0113] A pUC57 plasmid containing a T7 promoter (SEQ ID NO: 68), a 5'UTR (SEQ ID NO: 69), the coding sequence (CDS) of each reconstructed polypeptide, a 3'UTR (SEQ ID NO: 70), a polyadenylic acid tail (SEQ ID NO: 71), and a restriction endonuclease site (sequence: GAAGAGC) was constructed (construction service was provided by Nanjing GenScript Biotechnology Co., Ltd.), which was linearized and purified to obtain a high-quality linearized plasmid as an in vitro transcription template. The linearized plasmid template was transcribed in vitro using T7 RNA polymerase, 3'-OMe-GAG cap analog, and an optimized transcription system and conditions to obtain capped mRNA. The capped mRNA was affinity purified by oligo dT and sterile filtered. The concentration and integrity of the obtained mRNA were analyzed by spectrophotometry (NanoDrop One, Thermo Scientific) and capillary electrophoresis (Agilent 5200). The capping rate and polyadenylation tail of mRNA were analyzed by liquid chromatography-mass spectrometry (LC-MS). The pH value, residual DNA, protein and double-stranded RNA impurities of the mRNA solution were also analyzed. Figure 2 A schematic diagram showing the composition of mRNA.

[0114] mRNA was encapsulated using LNPs formulated with compound 5 from patent CN118084714B, DSPC, cholesterol, and DMG-PEG2000. The mRNA concentration, encapsulation efficiency, mRNA integrity, LNP particle size, and Zeta potential of the mRNA-LNP samples were analyzed using Quant-iTTM RiboGreenTM RNA Assay Kit (Invitrogen)-microplate reader (Varioskan LUX, Thermo Scientific), capillary electrophoresis (Agilent 5200), and light scattering instrument (ZetasizerUltra). The pH value, endotoxin, and bioburden of the mRNA-LNP samples were also analyzed to ensure that they were suitable for preclinical research.

[0115] Using the above procedure, mRNA-LNPs of each reconstructed polypeptide were prepared as mRNA vaccines.

[0116] Example 3: Expression of mRNA-LNP encoding reconstructed polypeptide in HEK293 cells

[0117] Flow cytometry or enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of mRNA-LNP encoding the reconstructed polypeptide in HEK293 cells. Flow cytometry detection method: HEK293 cells were plated in a culture dish, and when the cells grew to 80% coverage, mRNA-LNP was added. After 24 hours of transfection, the cells were collected and the F protein on the cell surface was labeled with fluorescent antibodies that recognized the F protein IV epitope and the PreF protein Φ epitope (blank cells without mRNA transfection were used as negative controls). After incubation, the cells were washed and the fluorescence intensity was detected by flow cytometry. ELISA detection method: HEK293 cells were plated in culture dishes. When the cells grew to 80% coverage, mRNA-LNP was added. After 48 hours of transfection, the culture supernatant was collected, the precipitate was removed, and the supernatant was concentrated 15 times using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The concentration of PreF protein in the supernatant concentrate was then detected using an ELISA kit. The PreF content was detected based on the double antibody sandwich method using a capture monoclonal antibody that specifically recognizes and binds to the RSV PreF ø epitope and a detection monoclonal antibody-horseradish peroxidase (HRP) that recognizes the II / III epitope.

[0118] Figure 3 Representative ELISA test results encoding reconstructed polypeptides without transmembrane domains are shown. In the figure, cells transfected with mRNA-LNPs encoding PreF1-T4, PreF2-T4, PreF3-T4, PreF4-T4, PreF1-IZ, and PreF1-MTQ reconstructed polypeptides all expressed PreF protein in the cell culture supernatant. Figure 4 Representative flow cytometry results of remodeled peptides containing transmembrane domains labeled with PreFø epitope antibodies are shown. The figure shows cells transfected with PreF1-F / TM A 、PreF1-T4-F / TM A 、PreF2-F / TM A 、PreF2-T4-F / TM A 、PreF3-F / TM A 、PreF3-T4-F / TM A 、PreF1-IZ-F / TM A 、PreF1-MTQ-F / TM A , PreF1-T4-HA / TM reconstructed polypeptide mRNA-LNP, the cellular PreF protein positivity rate was above 98%. Figure 5 Representative flow cytometry results of remodeled peptides containing transmembrane domains labeled with F protein IV epitope antibodies are shown. The figure shows cells transfected with PreF1-T4-F / TM A525-550 、PreF4-F / TM B 、PreF4-T4-F / TMB , PreF1-T4-gp160 / TM, PreF1-T4-S / TM reconstructed polypeptide mRNA-LNP, the cellular PreF protein positive rate is between 17.4-99.3%. Among them, the cellular F protein positive rate and mean fluorescence intensity (MFI) of PreF1-T4-gp160 / TM and PreF1-T4-S / TM reconstructed polypeptides are lower than those of other reconstructed polypeptides, but significantly better than the negative control group without mRNA-LNP transfection. The cellular F protein positive rate of the negative control group without mRNA-LNP transfection is 0.12%. The MFI of PreF1-T4-gp160 / TM and PreF1-T4-S / TM reconstructed polypeptides is about twice that of the negative control group.

[0119] It should be noted that in this example, the flow cytometry test only detected the F protein on the cell surface, and the ELISA test only detected the protein secreted outside the cell. Therefore, the detection value does not represent the expression of the total protein, but only proves that all mRNA-LNPs can express and produce F protein.

[0120] In summary, the above results indicate that the reconstructed polypeptides of the present invention can be expressed in cells.

[0121] Example 4: Experimental Animal Immunization and Sample Collection

[0122] 6-8 week old BALB / c female mice were randomly divided into groups of 6, and each round of experiments included a blank control group in addition to the experimental group. On day 0, the mRNA-LNP vaccine was injected into the mice by intramuscular injection on the outside of the right hind leg of the mice. The injection dose was 5μg / mouse and the injection volume was 50μL / mouse. The blank control group was injected with only the same volume of normal saline. On day 14, booster immunization was performed in the same way with the same dose. The weight of the mice was monitored during the period to avoid abnormal conditions. On day 28, the eyeballs were removed to obtain whole blood and separate the serum.

[0123] Example 5: Plaque reduction neutralization test (FRNT) to detect the neutralizing antibody titer in the serum of immunized mice

[0124] The focus reduction neutralization test (FRNT) was used to detect the serum neutralizing antibody titer NT50. The F protein sequence of the vaccine was derived from the A-type strain and the neutralizing antibody was detected by RSV-A2-GFP virus, and the neutralizing antibody was detected by RSV-B1-GFP virus when it was derived from the B-type strain.

[0125] HEP-2 cells were plated in 96-well cell culture plates. When the cells grew to 95% coverage, subsequent experiments were started. Serum complement inactivation: The serum was pre-inactivated in a 56°C water bath for 30 minutes. Serum gradient dilution: The serum was diluted with diluent (DMEM medium + 10% FBS), with an initial dilution of 40 times, and then diluted in 3-fold multiples, for a total of 8 dilution gradients. Virus dilution: The F protein sequence of the vaccine was derived from the A-type strain (PreF1, PreF2, PreF3 related reconstructed polypeptides) and the neutralizing antibodies were detected using RSV-A2-GFP virus. It was derived from the B-type strain (PreF4 related reconstructed polypeptides) and the neutralizing antibodies were detected using RSV-B1-GFP virus. Dilute the virus stock solution to 10 4 pfu / mL. Virus neutralization: Take 220μL of serum dilution and virus dilution respectively, mix them evenly, and incubate them in a 37℃ incubator for 1h. Serum-virus mixture is added to HEP-2 cell 96-well plate: the cell culture supernatant is removed, and 200μL of neutralized serum-virus mixture is added to each well. Two replicates are made for each serum dilution, and the cell culture plate is placed in a 37℃ incubator for 48h. Fluorescent spot reading: remove the culture medium, wash the cell surface twice with PBS, pat dry the liquid, and use an enzyme-linked spot analyzer to read the lesion spot count results. Finally, the RSV neutralizing antibody titer is calculated based on the virus titer and serum dilution multiple.

[0126] The results and analysis are as follows:

[0127] 1) Neutralizing antibody titers induced by mRNA vaccines of the present invention based on different RSV F proteins

[0128] Figure 6 AD in the figure respectively shows the neutralizing antibody titer levels induced by the mRNA vaccine based on the reconstructed polypeptides of the present invention, PreF1, PreF2, PreF3, and PreF4, which shows that the neutralizing antibody levels induced by each PreF antigen containing both the trimerization domain T4 foldon and the transmembrane domain F / TM (i.e., the reconstructed polypeptide of the present invention) are higher than those containing only T4 or only F / TM (i.e., the control polypeptide). A The geometric mean titers (GMT) of induced neutralizing antibodies were PreF1-F / TM A , 2.88, 2.70 times of PreF1-T4. PreF2-T4-F / TM A The induced neutralizing antibody GMTs are PreF2-F / TM A , 1.77, 1.14 times of PreF2-T4. PreF3-T4-F / TM A The induced neutralizing antibody GMTs are PreF3-F / TM A, 6.91, 1.71 times of PreF3-T4. PreF4-T4-F / TM B The induced neutralizing antibody GMTs are PreF4-F / TM B , 2.04 and 1.43 times of PreF4-T4. This shows that the ability of the reconstructed polypeptide of the present invention to induce neutralizing antibodies is significantly better than that of the control polypeptide. This shows that connecting the transmembrane domain to the C-terminus of the trimerization domain can significantly improve its immunogenicity.

[0129] 2) Neutralizing antibody titers induced by mRNA vaccines of the reconstructed polypeptides of the present invention based on different trimerization domains

[0130] Figure 7 The neutralizing antibody titer levels induced by the mRNA vaccines of the reconstructed polypeptides of the present invention based on IZ or MTQ were shown, which showed that the mRNA vaccines of the reconstructed polypeptides of the present invention containing the trimerization domains IZ or MTQ all induced higher levels of neutralizing antibody titers, which indicates that the reconstructed polypeptides of the present invention containing different trimerization domains IZ or MTQ all induced higher neutralizing antibody titers; and they were all higher than the control mRNA vaccines containing only the trimerization domain but not the transmembrane domain, PreF1-IZ-F / TM A The induced neutralizing antibody GMT was 6.77 times that of PreF1-IZ and PreF1-MTQ-F / TM A The induced neutralizing antibody GMT was 1.66 times that of PreF1-MTQ, which indicates that the immunogenicity can be significantly improved by connecting the transmembrane domain to the C-terminus of the trimerization domain.

[0131] 3) Neutralizing antibody titers induced by mRNA vaccines of the present invention based on reconstructed polypeptides with different transmembrane domains

[0132] Figure 8 The neutralizing antibody titers induced by mRNA vaccines of the reconstructed polypeptides of the present invention with different transmembrane domains (homologous or heterologous) were shown, which showed that mRNA vaccines of the reconstructed polypeptides of the present invention with different transmembrane domains all induced higher levels of neutralizing antibody titers, which were higher than the corresponding control mRNA vaccines containing only the trimerization domain or only the transmembrane domain F / TM of the F protein itself. PreF1-T4-HA / TM, PreF1-T4-S / TM, PreF1-T4-gp160 / TM, PreF1-T4-F / TM A 、PreF1-T4-F / TM A525-550 The induced neutralizing antibody GMTs were 5.05, 2.43, 1.71, 2.70, and 3.66 times that of PreF1-T4, respectively. AThis indicates that the immunogenicity of the trimerization domain can be significantly improved by connecting the homologous or heterologous transmembrane domain to the C-terminus of the trimerization domain.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present invention.

Claims

1. A reconstructed polypeptide of respiratory syncytial virus (RSV) F protein, characterized in that: The RSV F protein reconstructed polypeptide includes the following domains directly connected to each other or connected by a linker from the N-terminus to the C-terminus: an extracellular domain, a trimerization domain and a transmembrane domain of the RSV F protein; wherein the trimerization domain is selected from: a minor fibrin fold of bacteriophage T4, MTQ, and an isoleucine zipper short chain; the trimerization domain insertion site is: a corresponding site at amino acid position 513 of the RSV F protein precursor polypeptide as shown in SEQ ID NO: 1-4; and the transmembrane domain is a homologous transmembrane domain or a heterologous transmembrane domain of the RSV F protein.

2. The RSV F protein reconstructed polypeptide according to claim 1, characterized in that The RSV F protein is the F protein of type A or type B RSV virus; And / or, the heterologous transmembrane domain is selected from: the transmembrane domain of HIV-1 virus gp160 protein, the transmembrane domain of influenza virus HA protein or the transmembrane domain of novel coronavirus S protein.

3. The RSV F protein reconstructed polypeptide according to claim 1, characterized in that The amino acid sequence of the trimerization domain is selected from the following: SEQ ID NO: 5, 6, 9; and / or, the amino acid sequence of the homologous transmembrane domain of the RSV F protein is shown in SEQ ID NO: 60, 72 or 73; and / or, the amino acid sequence of the heterologous transmembrane domain is selected from the group consisting of: SEQ ID NOs: 61-63; And / or, the C-terminus of the transmembrane domain further comprises the intracellular domain of the protein from which it is derived; And / or, the amino acid sequence of the linker is shown in SEQ ID NO:64 or SEQ ID NO:

65.

4. The RSV F protein reconstructed polypeptide according to claim 1, characterized in that The amino acid sequence of the RSV F protein reconstructed polypeptide is selected from the following: SEQ ID NO: 24, 27, 30, 33, 35, 37, 38, 39, 40, 74.

5. The RSV F protein reconstructed polypeptide according to claim 1, characterized in that The RSV F protein reconstructed polypeptide further comprises a signal peptide.

6. The RSV F protein reconstructed polypeptide according to claim 5, characterized in that The signal peptide is a homologous signal peptide or a heterologous signal peptide.

7. The RSV F protein reconstructed polypeptide according to claim 6, characterized in that The heterologous signal peptide is EV preM signal peptide, VSV-G signal peptide, IgE heavy chain signal peptide, IgK light chain signal peptide, Secrecon signal peptide, CD33 signal peptide or tissue-type plasminogen activator tPA signal peptide.

8. The RSV F protein reconstructed polypeptide according to claim 6, characterized in that The amino acid sequence of the homologous signal peptide is selected from the following: SEQ ID NO: 66-67; And / or, the amino acid sequence of the heterologous signal peptide is selected from the following: SEQ ID NO: 15-21.

9. A polynucleotide encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8.

10. The polynucleotide according to claim 9, characterized in that The polynucleotide is a DNA molecule or an mRNA molecule.

11. A nucleic acid construct comprising the polynucleotide according to claim 9 or 10, and at least one expression control element operably linked to the polynucleotide.

12. The nucleic acid construct according to claim 11, characterized in that The nucleic acid construct comprises a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75, or an RNA sequence corresponding to these sequences, or consists of them.

13. An expression vector comprising the nucleic acid construct according to claim 11 or 12.

14. A host cell transformed or transfected with the polynucleotide according to claim 9 or 10, the nucleic acid construct according to claim 11 or 12, or the expression vector according to claim 13.

15. A recombinant respiratory syncytial virus (RSV) antigen, characterized in that: The RSV recombinant antigen comprises a polymer of the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8.

16. The RSV recombinant antigen according to claim 15, characterized in that The RSV recombinant antigen comprises a trimer of the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8.

17. Use of the RSV F protein reconstructed polypeptide as described in any one of claims 1-8, the polynucleotide as described in claim 9 or 10, the nucleic acid construct as described in claim 11 or 12, the expression vector as described in claim 13, the host cell as described in claim 14, or the RSV recombinant antigen as described in claim 15 or 16 in the preparation of a vaccine for preventing respiratory syncytial virus infection.

18. The use according to claim 17, characterized in that The vaccine also contains other antigenic components.

19. A vaccine or immunogenic composition comprising a RSV F protein reconstructed polypeptide as described in any one of claims 1-8, a polynucleotide as described in claim 9 or 10, a nucleic acid construct as described in claim 11 or 12, an expression vector as described in claim 13, a host cell as described in claim 14, or an RSV recombinant antigen as described in claim 15 or 16, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

20. The vaccine or immunogenic composition according to claim 19, which is a respiratory syncytial virus recombinant protein vaccine, comprising the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8 or the RSV recombinant antigen according to claim 15 or 16 and an adjuvant.

21. The vaccine or immunogenic composition according to claim 20, characterized in that The adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, Matrix M2 adjuvant, GLA-SE adjuvant.

22. The vaccine or immunogenic composition according to claim 19, which is a respiratory syncytial virus DNA vaccine, comprising: (i) a eukaryotic expression vector comprising a DNA sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8; and (ii) DNA vaccine adjuvants.

23. The vaccine or immunogenic composition according to claim 22, characterized in that The DNA sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 7 is a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, and 75; And / or, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS or pcDNA series vectors; And / or, the DNA vaccine adjuvant is selected from: human granulocyte macrophage colony stimulating factor, human interleukin-12 and / or CpG oligonucleotide.

24. The vaccine or immunogenic composition according to claim 19, which is a respiratory syncytial virus mRNA vaccine, comprising: (I) an mRNA sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8; (II) mRNA vaccine adjuvants; and (III) a delivery carrier, wherein the delivery carrier is selected from one or a combination of lipid nanoparticles, cationic nanoemulsions, polypeptides, polymers, cationic polypeptide polymers, and cationic polypeptide lipid nanoparticles.

25. The vaccine or immunogenic composition according to claim 24, characterized in that The mRNA sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8 is an mRNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, and 75; And / or, the mRNA vaccine adjuvant is selected from: lipid nanoparticles, emulsions, Toll-like receptor agonists and / or CpG oligonucleotides.

26. The vaccine or immunogenic composition according to claim 19, which is a respiratory syncytial virus-viral vector vaccine, comprising: (1) Viral backbone vector; and (2) A DNA sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8, constructed into the viral backbone vector.

27. The vaccine or immunogenic composition according to claim 26, characterized in that The DNA sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8 is a DNA sequence as shown in one of SEQ ID NOs: 43, 46, 49, 52, 54, 56, 57, 58, 59, and 75; And / or, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

28. The vaccine or immunogenic composition according to claim 19, which is a respiratory syncytial virus nanoparticle vaccine, comprising the RSV F protein reconstructed polypeptide according to any one of claims 1-8 and a nanoparticle carrier.

29. The vaccine or immunogenic composition according to claim 28, characterized in that The nanoparticle carrier is ferritin, and the RSV F protein reconstructed polypeptide according to any one of claims 1 to 8 is covalently linked to ferritin and self-assembled into nanoparticles, so that the RSV F protein reconstructed polypeptide is presented on the surface of the nanoparticles.

30. The vaccine or immunogenic composition according to any one of claims 19 to 29, characterized in that The vaccine or immunogenic composition is in the form of a nasal spray, an oral formulation or a parenteral formulation.

31. The vaccine or immunogenic composition according to claim 30, characterized in that The nasal spray is selected from aerosols, sprays and powder sprays; and / or, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments; And / or, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or pushable preparation.

32. The method for preparing the RSV F protein reconstructed polypeptide according to any one of claims 1 to 4, characterized in that: The preparation method comprises: A nucleotide sequence encoding a signal peptide is added to the 5' end of the nucleotide sequence encoding the RSV F protein reconstructed polypeptide according to any one of claims 1 to 4 and the codon-optimized nucleotide sequence, and a nucleotide sequence of a histidine tag and a stop codon are added to the 3' end, cloned and expressed, the correct recombinant is screened, and then transfected into expression system cells for expression, the cell culture supernatant is collected, and the RSV F protein reconstructed polypeptide is isolated therefrom.

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