Respiratory syncytial virus reconstructed polypeptide and use thereof

Abstract

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

Description

Respiratory syncytial virus reconstructed peptides and their applications

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 202411660083.7, filed on November 20, 2024, entitled “Reconstructed Peptide of Respiratory Syncytial Virus and Its Application Thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of vaccine technology, specifically to a reconstructed respiratory syncytial virus polypeptide, a recombinant respiratory syncytial virus antigen formed therefrom, an immunogenic composition containing therefrom, and methods for preparing and using the same. Background Technology

[0004] 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 global demand for a safe and effective RSV vaccine.

[0005] The RSV fusion protein (F protein) is a key mediator of RSV viral particle fusion with host cells. It exhibits extremely high immunogenicity in its pre-fusion state 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.

[0006] Extensive research has been conducted on the conformational stability of RSV F proteins before fusion, but most studies focus on mutating amino acids at specific sites in the RSV F protein or introducing a trimer domain at the C-terminus of the F protein. Previous studies have introduced a trimer domain at the C-terminus of the F protein by replacing the transmembrane domain (TM) with a trimer domain. However, the impact of deleting the transmembrane domain after the introduction of the trimer domain on vaccine efficacy has not been thoroughly investigated. Summary of the Invention

[0007] This application provides a reconstructed polypeptide based on the pre-fusion F protein of respiratory syncytial virus (RSV), a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a host cell transformed or transfected with the polynucleotide, the nucleic acid construct, or the expression vector, a stabilized multimer formed by the reconstructed polypeptide, an immunogenic composition comprising any of the foregoing, and its use in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection.

[0008] Specifically, this application provides the following technical solutions:

[0009] In a first aspect, this application provides a respiratory syncytial virus (RSV) reconstructed polypeptide, wherein the RSV F protein reconstructed polypeptide comprises, from the N-terminus to the C-terminus, the following domains that are directly connected to each other or connected by linkers: an extracellular domain, a trimerization domain, and a transmembrane domain of the RSV F protein; wherein the trimerization domain insertion site is the corresponding site at amino acid position 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.

[0010] For the aforementioned RSVF protein reconstructed polypeptide, the RSV F protein can be the F0 protein derived from any RSV subtype or RSV strain, for example, it can be the wild-type F protein of RSVA2 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 NO: 1-4.

[0011] Furthermore, 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 retains the pre-fusion conformation of the F protein, for example, it can be a PreF mutant described 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 RSVF protein is an F protein mutant with at least 80% amino acid sequence identity with the corresponding wild-type F protein.

[0012] In a feasible implementation, the RSV F protein is the F protein of type A or type B RSV virus, or a mutant having at least 80% sequence identity with it.

[0013] In a feasible implementation, the trimerization domain is selected from:

[0014] Phage T4 minor fibrin foldon and its variants (e.g., sequences shown in SEQ ID NO: 5), MTQ and its variants (e.g., sequences shown in SEQ ID NO: 6), heptapeptide ideal triple coiled helix and its variants (e.g., sequences shown in SEQ ID NO: 7), isoleucine zipper (IZ) long chain and its variants (e.g., sequences shown in SEQ ID NO: 8), IZ short chain and its variants (e.g., sequences shown in SEQ ID NO: 9), leucine zipper GCN4 and its variants (e.g., sequences shown in SEQ ID NO: 10), pulmonary surfactant-associated protein D trimer domain and its variants (e.g., sequences shown in SEQ ID NO: 11), collagen trimer domain and its variants (e.g., sequences shown in SEQ ID NO: 12), cartilage matrix protein-1 long chain trimer domain and its variants (e.g., sequences shown in SEQ ID NO: 13) or cartilage matrix protein-1 short chain trimer domain and its variants (e.g., sequences shown in SEQ ID NO: 13). NO: 14) or other trimerization domains and their variants.

[0015] In feasible implementations, the transmembrane domain of the RSV F protein is the homologous transmembrane domain F / TM of the F protein (e.g., whose sequence is shown in SEQ ID NO: 60, 72, 73, 83), or a heterologous transmembrane domain, such as the transmembrane domain gp160 / TM of the HIV-1 virus gp160 protein and its variants (e.g., as shown in SEQ ID NO: 61), the transmembrane domain HA / TM of the influenza virus HA protein and its variants (e.g., whose sequence is shown in SEQ ID NO: 62), the transmembrane domain S / TM of the novel coronavirus S protein and its variants (e.g., whose sequence is shown in SEQ ID NO: 63), or other transmembrane domains and their variants.

[0016] Furthermore, in feasible implementations, the C-terminus of the transmembrane domain also includes the intracellular domain of its source protein. For example, when the transmembrane domain is a homologous transmembrane domain of the RSVF protein, the C-terminus of the homologous transmembrane domain also includes the intracellular domain of the RSVF protein; when the transmembrane domain is a heterologous transmembrane domain, the C-terminus of the heterologous transmembrane domain also includes the intracellular domain of its source heterologous protein.

[0017] Furthermore, in feasible embodiments, the linker has an amino acid sequence as shown in SEQ ID NO: 64 or SEQ ID NO: 65.

[0018] In a preferred embodiment, the RSVF protein remodeling polypeptide has an amino acid sequence selected from the following:

[0019] SEQ ID NO: 24, 27, 30, 33, 35, 37, 38, 39, 40, 74.

[0020] In a feasible implementation, the RSVF protein reconstructing polypeptide further comprises a signal peptide, which may be a homologous signal peptide or a heterologous signal peptide; optionally, the heterologous signal peptide may be an EVpreM 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 plasminogen activator tPA signal peptide.

[0021] Preferably, the homologous signal peptide has an amino acid sequence as shown in SEQ ID NO: 66 or 67;

[0022] Preferably, the heterologous signal peptide has an amino acid sequence as shown in any one of SEQ ID NO: 15-21;

[0023] Preferably, the signal peptide is located at the N-terminus.

[0024] Secondly, this application provides a polynucleotide that encodes the RSV F protein reconstructed polypeptide as described in the first aspect above.

[0025] In a specific implementation, the polynucleotide can be DNA or mRNA; preferably, the polynucleotide is a nucleotide sequence optimized with human codons.

[0026] Thirdly, this application provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and at least one expression regulatory element operatively linked to the polynucleotide.

[0027] In some embodiments, the nucleic acid construct comprises, or consists of, a DNA sequence as shown in one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75 or an RNA sequence corresponding to these sequences.

[0028] Fourthly, this application provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0029] Fifthly, this application provides a host cell wherein the cell is transformed or transfected with the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0030] Optionally, the host cell is a mammalian cell, insect cell, yeast cell, or bacterial cell;

[0031] Further optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells;

[0032] Alternatively, the bacterial cells may be Escherichia coli cells.

[0033] In a sixth aspect, this application provides a recombinant respiratory syncytial virus (RSV) antigen, wherein the RSV recombinant antigen comprises a multimer, preferably a trimer, of the RSVF protein reconstructed polypeptide as described in the first aspect above.

[0034] In a seventh aspect, this application provides the use of 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 in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection.

[0035] In a feasible implementation, the vaccine may contain the RSVF 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 as the sole antigenic component.

[0036] In other feasible implementations, in addition to the RSVF 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 may also contain other antigenic components.

[0037] Eighthly, this application provides a vaccine or immunogenic composition comprising an RSVF protein reconstructed polypeptide as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a host cell as described in the fifth aspect above, or an RSV recombinant antigen as described in the sixth aspect above, and physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

[0038] In some preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus recombinant protein vaccine, comprising the RSV F protein reconstituted polypeptide as described in the first aspect above or the RSV recombinant antigen as described in the sixth aspect above, and an adjuvant;

[0039] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, Matrix M2 adjuvant, and GLA-SE adjuvant.

[0040] In some other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus DNA vaccine, comprising:

[0041] (i) a eukaryotic expression vector containing a DNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above; and

[0042] (ii) DNA vaccine adjuvants;

[0043] Optionally, the DNA sequence encoding the RSVF protein reconstructed polypeptide as described in the first aspect above is a DNA sequence as shown in one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

[0044] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors;

[0045] Optionally, the DNA vaccine adjuvant is selected from: human granulocyte-macrophage community-stimulating factor, human interleukin-12 and / or CpG oligonucleotides.

[0046] In some other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising:

[0047] (I) The mRNA sequence encoding the RSV F protein reconstructed polypeptide as described in the first aspect above;

[0048] (II) mRNA vaccine adjuvants; and

[0049] (III) Delivery carrier, wherein the delivery carrier is selected from one or more of lipid nanoparticles, cationic nanoemulsions, peptides, polymers, cationic peptide polymers, and cationic peptide lipid nanoparticles.

[0050] 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 shown in one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

[0051] Optionally, the mRNA vaccine adjuvant is selected from: lipid nanoparticles, emulsions, Toll-like receptor agonists and / or CpG oligonucleotides.

[0052] In some other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus-viral vector vaccine, comprising:

[0053] (1) Viral backbone vector; and

[0054] (2) Constructing a DNA sequence encoding the reconstructed peptide of RSV F protein as described in the first aspect above into the viral backbone vector;

[0055] Optionally, the DNA sequence encoding the RSVF protein reconstructed polypeptide as described in the first aspect above is a DNA sequence as shown in any one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

[0056] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0057] In some other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus nanoparticle vaccine, comprising the RSVF protein reconstructed polypeptide and nanoparticle carrier as described in the first aspect above.

[0058] Optionally, the nanoparticle carrier is ferritin, and the RSVF protein reconstructed peptide described in the first aspect above is covalently linked to ferritin and self-assembled into nanoparticles, so that the RSVF protein reconstructed peptide is present on the surface of the nanoparticles.

[0059] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, an oral formulation, or a parenteral formulation;

[0060] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0061] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0062] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

[0063] Ninthly, this application provides a method for preparing an RSV F protein reconstructed polypeptide as described in the first aspect above, characterized in that the preparation method includes:

[0064] The codon-optimized nucleotide sequence encoding the RSV F protein reconstructed polypeptide described in the first aspect above is coupled with a nucleotide sequence encoding a signal peptide at the 5' end and a nucleotide sequence encoding a histidine tag and a stop codon at the 3' end. The recombinants are then cloned and expressed, and the correct recombinants are screened. They are then transfected into expression system cells for expression. The cell culture supernatant is collected, and the RSV F protein reconstructed polypeptide is isolated from it.

[0065] In one 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.

[0066] Tenthly, this application provides a method for preventing and / or treating respiratory syncytial virus infection, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the following substances: RSV F protein reconstructed polypeptide as described in the first aspect above, polynucleotide as described in the second aspect above, nucleic acid construct as described in the third aspect above, expression vector as described in the fourth aspect above, host cell as described in the fifth aspect above, RSV recombinant antigen as described in the sixth aspect above, and / or vaccine or immunogenic composition as described in the eighth aspect above.

[0067] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the veterinarian's judgment, taking into account factors such as the type of dosage form, the method of administration, the age and weight of the subject, and the subject's symptoms. Beneficial effects

[0068] This application designs a reconstructed RSV F protein peptide by introducing a trimerization domain at a specific site in the pre-fusion F protein of RSV and linking a homologous or heterologous transmembrane domain of the RSV F protein to the C-terminus of the trimerization domain. Experiments demonstrate that the pre-fusion conformation of the reconstructed RSV F protein peptide is more stable, and the stability of the resulting trimerization protein is also enhanced, leading to stronger immunogenicity and a higher immune response. The reconstructed RSV F protein peptide exhibits excellent immunogenicity and can stimulate the body to produce high levels of neutralizing antibody titers, which is of great significance for the clinical treatment and prevention of respiratory syncytial virus (RSV).

[0069] Furthermore, this application experimentally demonstrates that combinations of different trimerization domains with transmembrane domains from different sources can induce high levels of neutralizing antibody titers. This indicates that the above design method does not depend on a specific RSV subtype or a specific F protein or its mutants, and can achieve high levels of neutralizing antibody titers in a variety of different mutants. In short, the RSVF protein reconstructed peptide of this application has broad-spectrum effectiveness. Attached Figure Description

[0070] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0071] Figure 1 shows a schematic diagram of the structure of the polypeptide described in Example 1.

[0072] Figure 2 shows a schematic diagram of the mRNA structure described in Example 2.

[0073] Figure 3 shows representative ELISA results of mRNA-LNP encoding the pre-fusion F protein (PreF) mutant as described in Example 3 in HEK293 cells.

[0074] Figure 4 shows the expression of mRNA-LNP encoding the pre-fusion F protein (PreF) mutant described in Example 3 in HEK293 cells using PreF. Representative flow cytometry results of epitope antibody labeling.

[0075] Figure 5 shows representative flow cytometry results of expression of mRNA-LNP encoding the pre-fusion F protein (PreF) mutant described in Example 3 in HEK293 cells, labeled with an antibody against the F protein IV epitope.

[0076] Figure 6 shows the immunogenicity of mRNA-LNPs encoding different PreF protein mutants containing the trimer domain of the phage T4 minor fibrin foldon, as described in Example 5, in mice; where AD shows the neutralizing antibody titers in the serum of mice immunized with mRNA-LNPs encoding different PreF mutants PreF1, PreF2, PreF3, and PreF4, respectively; the neutralizing antibody titers are presented as a bar chart of NT50 geometric mean titer (GMT), with the GMT value marked at the top of the bar chart, and the error bars representing the 95% CI (confidence interval).

[0077] Figure 7 shows the immunogenicity of mRNA-LNP encoding the PreF protein mutant containing the MTQ or isoleucine zipper (IZ) short-chain trimer domain, as described in Example 5, in mice; the figure shows the encoding PreF1-IZ and PreF1-IZ-F / TM. A PreF1-MTQ, PreF1-MTQ-F / TM A Neutralizing antibody titers in the serum of mice immunized with mRNA-LNP were calculated. Neutralizing antibody titers are presented as NT50 GMT bar charts, with GMT values ​​labeled at the top of the bars. Error bars represent 95% CI.

[0078] Figure 8 shows the immunogenicity of mRNA-LNP encoding PreF mutants containing different transmembrane domains (TM) described in Example 5 in mice; the figure shows the PreF protein mutants PreF1-T4-HA / TM, PreF1-T4-S / TM, PreF1-T4-gp160 / TM, and PreF1-T4-F / TM encoding TMs containing influenza HA or novel coronavirus S protein, HIV-1 gp160, or RSV F protein. A PreF1-T4-F / TM A525-550 And as a comparison, PreF1-T4 and PreF1-F / TM A Neutralizing antibody titers in the serum of mice immunized with mRNA-LNP were calculated. Neutralizing antibody titers are presented as a bar chart of NT50GMT, with GMT values ​​labeled at the top of the bars. Error bars represent 95% CI.

[0079] Figure 9 shows the immunogenicity of mRNA-LNP of the PreF protein mutants with different trimer domain positions described in Example 5 in mice; the figure shows PreF1-T4-F / TM. A PreF1 524 -T4-F / TM A525-550 PreF1-F / TM A -T4、PreF1-F / TM A514-560 Neutralizing antibody titers in the serum of mice immunized with mRNA-LNP of -T4 and PreF1-T4 as a control. Neutralizing antibody titers are presented as NT50 GMT bar charts, with GMT values ​​labeled at the top of the bars. Error bars represent 95% CI. Detailed Implementation

[0080] To better understand this application, definitions and explanations of relevant terms are provided below.

[0081] The term "peptide" refers to any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). "Peptide" applies to amino acid polymers, including naturally occurring and non-naturally occurring amino acid polymers, as well as artificial chemical mimics of the corresponding naturally occurring amino acids, where one or more amino acid residues are non-natural, such as corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimic incorporated into a peptide via an amide bond or an amide bond mimic. Peptides have an amino-terminal (N-terminus) and a carboxyl-terminal (C-terminus) end. "Peptide" is used interchangeably with "peptide" or "protein" and is used herein to refer to polymers of amino acid residues.

[0082] The term "F protein" or "F polypeptide" refers to a protein or polypeptide that has the whole or part of the amino acid sequence of RSV F protein.

[0083] RSV F protein, or fusion protein, is an RSV envelope glycoprotein that promotes the fusion of the RSV viral membrane and the host cell membrane, playing a crucial role in respiratory syncytial virus infection. In nature, RSV F protein is initially synthesized as a single polypeptide precursor of approximately 574 amino acids, 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 cleaved by proteolytic hydrolysis. The remaining F0 residues form a trimer through oligomerization and are cleaved by two conserved furin cleavage sequences (approximately positions 109 and 136 of F0; e.g., RARR). 109 (amino acid residues 106-109 of F0) and RKRR 136 At amino acid residues 133-136 of F0, proteolytic processing by cellular proteases produces two disulfide-linked fragments, F1 and F2. The smaller F2 fragment originates from the N-terminal portion of the F0 precursor and includes approximately amino acid residues 26-109 of F0. The larger F1 fragment comprises the C-terminal portion of the F0 precursor (approximately amino acid residues 137-574 of F0), including an extracellular / luminal region (approximately amino acid residues 137-529), a transmembrane domain (approximately amino acid residues 525-550), and a C-terminal cytoplasmic tail (approximately amino acid residues 551-574). The extracellular portion of the RSV F protein is the RSV F extracellular domain, which includes the F2 protein and the F1 extracellular domain.

[0084] RSV F proteins exhibit significant sequence conservation across RSV subtypes. For example, in the F0 precursor molecule, RSV subtypes A and B share 90% sequence identity, and each RSV subtype A and B shares 81% sequence identity with bovine RSV F protein. Within RSV subtypes, F0 sequence identity is even higher; for example, within each subtype of RSVA, B, and bovine subtypes, RSV F0 precursor proteins share approximately 98% sequence identity. Almost all identified RSV F0 precursor proteins are approximately 574 amino acids in length, with minor differences in length typically due to the length of the C-terminal cytoplasmic tail. Given the conservation of RSV F sequences, those skilled in the art can readily compare amino acid positions between different native RSV F sequences to identify corresponding RSVF amino acid positions between different RSV virus strains and subtypes. Therefore, the conservation of RSV F protein sequences across strains and subtypes allows for the comparison of amino acids at specific positions within RSV F proteins using reference RSV F sequences. Unless the context otherwise requires, the amino acid substitutions disclosed herein are numbered with reference to the RSVF0 polypeptide sequences shown in SEQ ID NO: 1-4.

[0085] The three F2-F1 promeromers oligomerize in the mature F protein, adopting a metastable pre-fusion conformation. Upon contact with the target cell membrane, they undergo a conformational change to the post-fusion conformation. This conformational change exposes a hydrophobic sequence called the fusion peptide, located at the N-terminus of the F1 extracellular domain. This peptide binds to the host cell membrane and promotes the fusion of the viral or infected cell membrane with the target cell membrane.

[0086] A "domain" or "structural domain" of a polypeptide or protein refers to a structurally defined element within the polypeptide or protein. For example, a "trimerizing domain" is an amino acid sequence within the polypeptide that promotes the assembly of the polypeptide into a trimer. For instance, a trimerizing domain can promote the assembly into a trimer via association with other trimerizing domains (of other polypeptides having the same or different amino acid sequences). A "transmembrane domain" is an amino acid sequence that can insert into a lipid bilayer (e.g., the lipid bilayer of a cell, virus, or virus-like particle) or anchor an antigen to the membrane. This term is also used to refer to polynucleotides encoding such peptides or polypeptides. The aforementioned structural domains in the RSVF protein reconstructed polypeptide described in this application can be directly linked or linked through natural or artificial linkers, adapters, or spacer sequences.

[0087] The terms "linker," "connector," "spacer," or "spacer sequence" refer to a natural or artificial bifunctional molecule that can be used to link two molecules into a continuous molecule. Non-limiting examples of peptide linkers include glycine-serine peptide linkers and fragments of any length between amino acids 514-524 of the RSV F protein. Unless the context otherwise requires, references to "linking" a first polypeptide and a second polypeptide, or to "linking" two polypeptides together, or to a first polypeptide having a "link" with a second polypeptide, refer to a covalent connection via peptide bonds (e.g., via a peptide linker) such that the first and second polypeptides form a continuous polypeptide chain. If a peptide linker is involved, the covalent connection between the first and second polypeptides can be to the N-terminus and C-terminus of the peptide linker. The term "mutant" or "variant" refers to a molecule whose amino acid sequence differs from the 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 the natural or reference sequence. Typically, variants share at least 50% homology with the natural or reference sequence. In some implementations, the variant shares at least 80% or at least 90% homology with the natural or reference sequence.

[0088] The term "homology" refers to the overall correlation between aggregate molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Aggregates (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a critical level of similarity or identity as determined by the alignment of matching residues are said to be homologous. 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 compared sequences. In some embodiments, aggregate molecules are considered "homological" if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homology" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if they encode polypeptides that are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical with respect to at least one extension having at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by their ability to encode an extension having at least 4 to 5 uniquely defined amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode an extension having at least 4 to 5 uniquely defined amino acids. Two protein sequences are considered homologous if they are at least 50%, 60%, 70%, 80%, or 90% identical with respect to at least one extension having at least 20 amino acids. The terms "polynucleotide" and "nucleic acid sequence" refer to a polymeric form of nucleotides with a length of at least 10 bases. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of any nucleotide. The 5' and 3' orientations of nucleic acids are defined by reference to the connectivity of individual nucleotide units and assigned according to the carbon positions of the deoxyribose (or ribose) sugar ring. The information (encoding) of a polynucleotide sequence is read in the 5' to 3' orientations.

[0089] The term "reconstructed polypeptide," or "recombinant polypeptide," refers to a polypeptide that has a non-naturally occurring sequence or a sequence generated by artificially combining two or more sequence segments that are otherwise separate, which may be in the same protein or in different proteins.

[0090] In some embodiments, the nucleotide vaccine of this disclosure comprises at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one respiratory syncytial virus (RSV) antigenic polypeptide, wherein said RNA comprises at least one chemical modification.

[0091] The terms “chemically modified” and “chemically modified” refer to modifications of at least one of the ribonucleosides or deoxyribonucleosides of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C), in terms of their position, pattern, percentage, or population. Generally, these terms do not refer to ribonucleotide modifications of the naturally occurring 5' mRNA cap portion. Modifications of polynucleotides include, but are not limited to, those described herein, and include (but are not explicitly limited to) those involving chemical modifications. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may contain naturally occurring, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may include any applicable modifications to, for example, sugars, nucleotide bases, or nucleoside linkages (e.g., linkages to phosphate esters, phosphodiester linkages, or the phosphodiester backbone).

[0092] The term "heterogeneous" refers to nucleic acids, peptides, or other cellular components that are not typically found in nature and / or originate from different sources or species.

[0093] "Antigen" refers to a compound, composition, or substance that can stimulate antibody production and / or T cell response in an animal, including compositions introduced into an animal by injection, adsorption, or other means. The term "antigen" includes all relevant antigenic epitopes. The term "epitaph" or "antigenic determinant" refers to a site on an antigen that elicits a B and / or T cell response. "Dominant antigenic epitope" or "dominant epitope" refers to those epitopes that elicit a functionally significant host immune response (e.g., antibody response or T cell response). Thus, regarding a protective immune response against a pathogen, a dominant antigenic epitope refers to those antigenic modules that, when recognized by the host immune system, produce protection against disease caused by that pathogen. The term "T cell epitope" refers to an epitope that is specifically bound by T cells (via T cell receptors) when it binds to a suitable MHC molecule. "B cell epitope" refers to an epitope that is specifically bound by an antibody (or B cell receptor molecule).

[0094] A “vaccine” is a preparation of an immunogenic substance that can stimulate an immune response and is administered to prevent, improve, or treat an infectious disease or other type of illness. Immunogenic substances may include attenuated or killed microorganisms (e.g., bacteria or viruses), or antigenic proteins, peptides, or DNA derived from them. Vaccines may include exposed immunogens (e.g., recombinant RSVF extracellular domain trimers or nucleic acid molecules encoding them), viruses, cells, or one or more cellular components. Vaccines can elicit both preventative (protective) and therapeutic responses. Methods of administration vary depending on the vaccine but may include inoculation, ingestion, inhalation, or other forms of administration. Vaccines may be administered with adjuvants to enhance the immune response. In a specific, non-limiting example, a vaccine prevents and / or reduces the severity of symptoms associated with RSV infection and / or reduces viral load compared to a control.

[0095] "Carrier" or "excipient" refers to pharmaceutically acceptable excipients, specifically excipients and additives used in the production of drugs and the formulation of prescriptions. These are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a carrier and improving stability, pharmaceutically acceptable excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of drugs. Based on their function and use, pharmaceutical excipients can be classified into solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0096] "Recombinant" is a molecule having a sequence that is not naturally occurring, such as comprising one or more nucleic acid substitutions, deletions, or insertions, and / or having a sequence prepared by an artificial combination of two otherwise separated sequence segments. This artificial combination can be accomplished by chemical synthesis or, more commonly, by artificial manipulation of the separated nucleic acid segments (e.g., by genetic engineering techniques). A recombinant protein is a protein having a sequence that is not naturally occurring or having a sequence prepared by an artificial combination of two otherwise separated 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 bacterium or eukaryotic cell) or into the genome of a recombinant virus.

[0097] "Expression regulatory element" refers to a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is operatively linked. An expression control sequence is operatively linked to a nucleic acid sequence when it controls and regulates transcription and (appropriately) translation of the nucleic acid sequence. Therefore, an expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, intron splicing signals (maintaining the correct reading frame of the gene to allow for correct translation of mRNA), and stop codon. The term "regulatory element" is intended to include at least the component whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion coupler sequences. An expression regulatory element may include a promoter.

[0098] A “promoter” is the smallest sequence sufficient to direct transcription. This includes the T7 promoter, as well as promoter elements sufficient to make promoter-dependent gene expression controllable with respect to cell type specificity, tissue specificity, or induced by external signals or reagents; these elements can be located at the 5' or 3' of the gene. This includes constitutive and inducible promoters (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac heterozygous promoters) of bacteriophage λ can be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the mammalian cell genome (such as metallothionein promoters) or promoters derived from mammalian viruses (such as retroviral long terminal repeat sequences; adenovirus late promoters; vaccinia virus 7.5K promoters) can be used. Promoters generated through recombinant DNA or synthetic techniques can also be used to provide nucleic acid sequences for transcription.

[0099] "5'UTR" refers to the non-coding polypeptide mRNA region located directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by ribosomes). "3'UTR" refers to the non-coding polypeptide mRNA region located directly downstream (i.e., 3') of the stop codon (i.e., the codon of the mRNA transcript that transmits the translation termination signal).

[0100] "Coded sequence CDS" is a continuous extension of RNA that begins with a start codon (e.g., methionine (AUG)) and ends with a stop codon (e.g., UAA, UAG, or UGA) and encodes a polypeptide.

[0101] A "polyA tail," or "polyadenylated tail," is located downstream of the 3' UTR in mRNA and contains multiple consecutive adenosine monophosphate (ATP). A polyA tail can contain 10 to 300 ATP. For example, a 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 ATP. In some embodiments, the polyA tail contains 50 to 250 ATP. In some embodiments, the polyA tail contains non-adenosine nucleotides that segment the consecutive ATP. In relevant biological contexts (e.g., in cells, in vivo), the poly(A) tail serves to protect mRNA from enzymatic degradation, such as in the cytoplasm, and facilitates transcription termination and / or mRNA export from the nucleus and translation.

[0102] "Host cell" refers to a cell in which the vector can proliferate and express the vector's nucleic acid. The cell can be prokaryotic or eukaryotic. The term also includes any offspring of the target host cell. It should be understood that all offspring may differ from the parent cell because mutations can occur during replication. However, when the term "host cell" is used, such offspring are included.

[0103] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0104] Furthermore, to better illustrate this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0105] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0106] Example 1: Structural Design of Reconstructed Peptides

[0107] In this embodiment, the following reconstructed polypeptides of this application and their control polypeptides were designed respectively.

[0108] This application reconstructs the polypeptide:

[0109] PreF1-T4-F / TM A (its sequence is shown in SEQ ID NO: 24), PreF2-T4-F / TM A (its sequence is shown in SEQ ID NO: 27), PreF3-T4-F / TM A (its sequence is shown in SEQ ID NO: 30), PreF4-T4-F / TM B (its sequence is shown in SEQ ID NO: 33), PreF1-MTQ-F / TM A (its sequence is shown in SEQ ID NO: 35), PreF1-IZ-F / TM A (The sequence is shown in SEQ ID NO: 37), PreF1-T4-gP160 / TM (The sequence is shown in SEQ ID NO: 38), PreF1-T4-HA / TM (The sequence is shown in SEQ ID NO: 39), PreF1-T4-S / TM (The sequence is shown in SEQ ID NO: 40), PreF1-T4-F / TM A525-550 (Its sequence is shown in SEQ ID NO: 74).

[0110] Control peptide:

[0111] PreF1-F / TM A (its sequence is shown in SEQ ID NO: 22), PreF1-T4 (its sequence is shown in SEQ ID NO: 23), PreF2-F / TM A (its sequence is shown in SEQ ID NO: 25), PreF2-T4 (its sequence is shown in SEQ ID NO: 26), PreF3-F / TM A (its sequence is shown in SEQ ID NO: 28), PreF3-T4 (its sequence is shown in SEQ ID NO: 29), PreF4-F / TM B (The sequence is shown in SEQ ID NO: 31), PreF4-T4 (The sequence is shown in SEQ ID NO: 32), PreF1-MTQ (The sequence is shown in SEQ ID NO: 34), PreF1-IZ (The sequence is shown in SEQ ID NO: 36), PreF1-F / TM A514-560 -T4 (its sequence is shown in SEQ ID NO: 82), PreF1-F / TM A-T4 (its sequence is shown in SEQ ID NO: 79), PreF1 524 -T4-F / TM A525-550 (Its sequence is shown in SEQ ID NO: 77).

[0112] The sequences of the above-mentioned polypeptides are all sequences that do not contain their endogenous signal peptides, and the endogenous signal peptide sequences used are shown in SEQ ID NO: 66 or 67; among them, PreF1-F / TM A (its sequence is shown in SEQ ID NO: 22), PreF1-T4 (its sequence is shown in SEQ ID NO: 23), PreF1-T4-F / TM A (its sequence is shown in SEQ ID NO: 24), PreF2-F / TM A (its sequence is shown in SEQ ID NO: 25), PreF2-T4 (its sequence is shown in SEQ ID NO: 26), PreF2-T4-F / TM A (its sequence is shown in SEQ ID NO: 27), PreF3-F / TM A (its sequence is shown in SEQ ID NO: 28), PreF3-T4 (its sequence is shown in SEQ ID NO: 29), PreF3-T4-F / TM A (its sequence is shown in SEQ ID NO: 30), PreF1-MTQ (its sequence is shown in SEQ ID NO: 34), PreF1-MTQ-F / TM A (its sequence is shown in SEQ ID NO: 35), PreF1-IZ (its sequence is shown in SEQ ID NO: 36), PreF1-IZ-F / TM A (The sequence is shown in SEQ ID NO: 37), PreF1-T4-gP160 / TM (The sequence is shown in SEQ ID NO: 38), PreF1-T4-HA / TM (The sequence is shown in SEQ ID NO: 39), PreF1-T4-S / TM (The sequence is shown in SEQ ID NO: 40), PreF1-T4-F / TM A525-550 (its sequence is shown in SEQ ID NO: 74), PreF1-F / TM A514-560 -T4 (its sequence is shown in SEQ ID NO: 82), PreF1-F / TM A -T4 (its sequence is shown in SEQ ID NO: 79), PreF1 524 -T4-F / TM A525-550The endogenous signal peptide sequence of (its sequence is shown in SEQ ID NO: 77) is shown in SEQ ID NO: 66, and the endogenous signal peptide sequences of the remaining polypeptides are shown in SEQ ID NO: 67.

[0113] The structural diagrams of the above-mentioned polypeptides are shown in Figure 1.

[0114] Example 2: Preparation of reconstructed peptide mRNA and packaging of lipid nanoparticles (LNPs)

[0115] A pUC57 plasmid containing the T7 promoter (SEQ ID NO: 68), 5'UTR (SEQ ID NO: 69), coding sequences (CDS) of each reconstructed polypeptide, 3'UTR (SEQ ID NO: 70), polyadenylate tail (SEQ ID NO: 71), and restriction endonuclease site (sequence GAAGAGC) was constructed (construction service provided by Nanjing Genscript Biotech Co., Ltd.). This plasmid was linearized and purified by enzyme digestion to obtain a high-quality linearized plasmid, which served as a template for in vitro transcription. The linearized plasmid template was transcribed in vitro using T7 RNA polymerase, a 3'-OMe-GAG cap analog, and optimized transcription systems and conditions to obtain capped mRNA. The capped mRNA was purified by oligodT affinity chromatography and sterile filtration. 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 polyadenylate 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 shows a schematic diagram of the mRNA composition.

[0116] 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 size, and zeta potential of the mRNA-LNP samples were analyzed using the Quant-iT™ RiboGreen™ RNAAssay Kit (Invitrogen)-ELISA reader (Varioskan LUX, Thermo Scientific), capillary electrophoresis (Agilent 5200), and light scattering instrument (Zetasizer Ultra). The pH, endotoxin, and bioburden of the mRNA-LNP samples were also analyzed to ensure suitability for preclinical studies.

[0117] Using the above procedure, mRNA-LNPs of each reconstructed polypeptide were prepared as mRNA vaccines.

[0118] Example 3: Expression of mRNA-LNP encoding the reconstructed polypeptide in HEK293 cells

[0119] The expression of mRNA-LNP encoding the reconstituted polypeptide in HEK293 cells was detected using flow cytometry or enzyme-linked immunosorbent assay (ELISA). Flow cytometry: HEK293 cells were seeded in culture dishes. When the cells reached 80% confluence, mRNA-LNP was added. After 24 hours of transfection, cells were collected, and the F protein on the cell surface was labeled with fluorescent antibodies recognizing epitopes IV and Φ of the PreF protein (blank cells without mRNA transfection served as a negative control). After incubation, the cells were washed, and the fluorescence intensity was detected by flow cytometry. ELISA: HEK293 cells were seeded in culture dishes. When the cells reached 80% confluence, mRNA-LNP was added. After 48 hours of transfection, the culture supernatant was collected, the precipitate was removed, and the supernatant was concentrated 15-fold using a 10kDa ultrafiltration tube. The concentration of PreF protein in the supernatant concentrate was then detected using an ELISA kit. The concentration of PreF protein was determined by specifically recognizing and binding to RSVPreF. Epitope capture monoclonal antibody and detection of recognition of II / III epitopes: Monoclonal antibody-horseradish peroxidase (HRP) based on double antibody sandwich method to detect PreF content.

[0120] Figure 3 shows representative ELISA results for reconstructed peptides lacking transmembrane domains. Cells transfected with mRNA-LNPs encoding the reconstructed peptides PreF1-T4, PreF2-T4, PreF3-T4, PreF4-T4, PreF1-IZ, and PreF1-MTQ all expressed PreF protein in their cell culture supernatants. Figure 4 shows the results of PreF protein expression for reconstructed peptides containing transmembrane domains. Representative flow cytometry results of epitope antibody labeling. The figure shows the cells transfected with the PreF1-F / TM antibody. 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 AThe mRNA-LNP of the PreF1-T4-HA / TM reconstructed peptide showed a PreF protein positivity rate of over 98%. Figure 5 shows representative flow cytometry results of the reconstructed peptide containing the transmembrane domain labeled with an antibody against the F protein IV epitope. The figure shows cells transfected with the protein encoding PreF1-T4-F / TM. A525-550 PreF4-F / TM B PreF4-T4-F / TM B The mRNA-LNP positivity rates of the PreF protein in cells of the PreF1-T4-gp160 / TM and PreF1-T4-S / TM reconstructed peptides ranged from 17.4% to 99.3%. While the positivity rates and mean fluorescence intensity (MFI) of the PreF1-T4-gp160 / TM and PreF1-T4-S / TM reconstructed peptides were lower than those of other reconstructed peptides, they were significantly better than those of the negative control group without mRNA-LNP transfection. The positivity rate of F protein in the negative control group without mRNA-LNP transfection was 0.12%. The MFI of the PreF1-T4-gp160 / TM and PreF1-T4-S / TM reconstructed peptides was approximately twice that of the negative control group.

[0121] It should be noted that in this embodiment, the flow cytometry assay only detected F protein on the cell surface, and the ELISA assay only detected proteins secreted outside the cell. Therefore, the detection values ​​do not represent the total protein expression, but only prove that all mRNA-LNPs can express and produce F protein.

[0122] In summary, the above results demonstrate that the reconstructed peptides of this application can be expressed in cells.

[0123] Example 4: Immunization and Sample Collection of Laboratory Animals

[0124] Six- to eight-week-old BALB / c female mice were randomly assigned to groups of six. Each round of experiments included both experimental and control groups. On day 0, the mRNA-LNP vaccine was injected intramuscularly into the lateral aspect of the right hind leg of each mouse at a dose of 5 μg / mouse and an injection volume of 50 μL / mouse. The control group received only the same volume of saline. On day 14, a booster immunization was administered in the same manner and at the same dose. Mouse weight was monitored throughout the period to prevent any abnormalities. On day 28, whole blood was collected by enucleation, and serum was separated.

[0125] Example 5: Plaque Reduction Neutralization Assay (FRNT) for detecting neutralizing antibody titers in the serum of immunized mice

[0126] Serum neutralizing antibody titers (NT50) were detected using the focal reduction neutralization assay (FRNT). Neutralizing antibodies against the F protein sequence of the vaccine derived from the type A strain were detected using RSV-A2-GFP virus, while those derived from the type B strain were detected using RSV-B1-GFP virus.

[0127] HEP-2 cells were seeded into 96-well cell culture plates. When the cells reached 95% confluence, subsequent experiments began. Serum complement inactivation: Serum was incubated at 56°C for 30 min to inactivate complement. Serum serial dilution: Serum was diluted with diluent (DMEM medium + 10% FBS) starting at a 40-fold dilution, followed by 3-fold serial dilutions for a total of 8 dilution gradients. Virus dilution: The F protein sequence of the vaccine was derived from type A strains (PreF1, PreF2, PreF3 related reconstructed peptides) and neutralizing antibodies were detected using RSV-A2-GFP virus. The sequence was derived from type B strains (PreF4 related reconstructed peptides) and neutralizing antibodies were detected using RSV-B1-GFP virus. The original virus stock solution was diluted to 10... 4 pfu / mL. Virus neutralization: Mix 220 μL of serum diluent and virus diluent separately and incubate at 37°C for 1 h. Add serum-virus mixture to HEP-2 cell 96-well plates: Remove cell culture supernatant, add 200 μL of the neutralized serum-virus mixture to each well, perform two replicates for each serum dilution, and incubate the cell culture plate at 37°C for 48 h. Fluorescent spot reading: Remove culture medium, wash the cell surface twice with PBS, pat dry, and read the spot count using an ELISA reader. Finally, combine the virus titer and serum dilution factor to calculate the RSV neutralizing antibody titer.

[0128] The results and analysis are as follows:

[0129] 1) Neutralizing antibody titers induced by mRNA vaccines based on reconstructed peptides of this application using different RSVF proteins.

[0130] Figure 6 (AD) shows the neutralizing antibody titer levels induced by mRNA vaccines based on the reconstructed peptides of this application (PreF1, PreF2, PreF3, and PreF4), respectively. It indicates that the neutralizing antibody levels induced by each PreF antigen (i.e., the reconstructed peptides of this application) containing both the trimerization domain T4 foldon and the transmembrane domain F / TM are higher than those induced by antigens containing only T4 or only F / TM (i.e., the control peptides). PreF1-T4-F / TM A The induced geometric mean titers (GMT) of the neutralizing antibodies were PreF1-F / TM. A 2.88 and 2.70 times that of PreF1-T4. PreF2-T4-F / TM AThe induced neutralizing antibody GMTs were PreF2-F / TM. A 1.77 and 1.14 times that of PreF2-T4. PreF3-T4-F / TM A The induced neutralizing antibody GMTs are PreF3-F / TM. A 6.91 times and 1.71 times that of PreF3-T4. PreF4-T4-F / TM B The induced neutralizing antibody GMTs were PreF4-F / TM. B The levels of PreF4-T4 and PreF4-T4 were 2.04 and 1.43 times higher, respectively. This indicates that the ability of the reconstructed peptides in this application to induce neutralizing antibodies is significantly superior to that of the control peptides. This also demonstrates that linking the transmembrane domain to the C-terminus of the trimerization domain can significantly improve its immunogenicity.

[0131] 2) Neutralizing antibody titers induced by mRNA vaccines containing reconstructed peptides of this application based on different trimerization domains.

[0132] Figure 7 shows the neutralizing antibody titer levels induced by mRNA vaccines containing the reconstructed peptides of this application based on IZ or MTQ. It indicates that all mRNA vaccines containing either the trimerizing domain IZ or MTQ induced high levels of neutralizing antibody titers. This demonstrates that all reconstructed peptides of this application containing different trimerizing domains IZ or MTQ induced high neutralizing antibody titers; furthermore, these titers were higher than those of the control mRNA vaccine containing only the trimerizing domain and lacking the transmembrane domain. PreF1-IZ-F / TM A The induced neutralizing antibody GMT was 6.77 times that of PreF1-IZ, PreF1-MTQ-F / TM A The induced neutralizing antibody GMT was 1.66 times that of PreF1-MTQ, indicating that its immunogenicity can be significantly improved by linking the transmembrane domain to the C-terminus of the trimerization domain.

[0133] 3) Neutralizing antibody titers induced by mRNA vaccines based on reconstructed peptides of this application with different transmembrane domains.

[0134] Figure 8 shows the neutralizing antibody titers induced by mRNA vaccines containing different transmembrane domains (homologous or heterologous). It demonstrates that the mRNA vaccines containing different transmembrane domains induced high levels of neutralizing antibody titers, all higher than the corresponding control mRNA vaccines containing only the trimer domain or only the F protein's own transmembrane domain F / TM. PreF1-T4-HA / TM, PreF1-T4-S / TM, PreF1-T4-gp160 / TM, PreF1-T4-F / TM A PreF1-T4-F / TM A525-550The induced neutralizing antibody GMTs were 5.05, 2.43, 1.71, 2.70, and 3.66 times that of PreF1-T4, respectively, and were respectively the values ​​of PreF1-F / TM. A The immunogenicity was 5.39, 2.60, 1.82, 2.88, and 3.91 times higher than that of the trimerization domain. This indicates that by linking the homologous or heterologous transmembrane domain to the C-terminus of the trimerization domain, its immunogenicity can be significantly improved.

[0135] 4) Neutralizing antibody titers induced by mRNA vaccines of reconstructed peptides with different trimerization domain positions

[0136] Figure 9 shows the neutralizing antibody titers induced by mRNA vaccines containing reconstructed peptides with trimer domains introduced at different positions and by the mRNA vaccine containing the reconstructed peptide of this application. It demonstrates that different trimer domain positions affect the neutralizing antibody titer, and the reconstructed peptide mRNA vaccine of this application induced a higher level of neutralizing antibody titer. PreF1-T4-F / TM A The induced neutralizing antibody GMT is PreF1 524 -T4-F / TM A525-550 PreF1-F / TM A -T4、PreF1-F / TM A514-560 -T4 was 1.63-fold, 1.60-fold, and 2.36-fold higher. This indicates that introducing a trimerizing domain at position 513 of the F1 protein and linking the transmembrane domain to the C-terminus of the trimerizing domain can significantly improve its immunogenicity.

[0137] The sequences involved in this application are as follows:

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of this application. Industrial applicability

[0139] The respiratory syncytial virus (RSV) reconstructed peptide provided in this application introduces a trimerization domain at a specific site in the RSV F protein before RSV fusion, and links a homologous or heterologous transmembrane domain of the RSV F protein to the C-terminus of the trimerization domain. Experiments have demonstrated that the pre-fusion conformation of the RSV F protein reconstructed peptide of this application is more stable, and the stability of the resulting trimerization protein is also enhanced, thereby exhibiting stronger immunogenicity and a higher immune response. The RSV F protein reconstructed peptide of this application possesses excellent immunogenicity and can stimulate the body to produce high levels of neutralizing antibody titers, which is of great significance for the clinical treatment and prevention of respiratory syncytial virus.

Claims

1. A reconstructed polypeptide of respiratory syncytial virus (RSV) F protein, characterized in that, The RSV F protein reconstructed polypeptide comprises, from the N-terminus to the C-terminus, the following domains that are directly connected to each other or connected by linkers: the extracellular domain, trimerization domain, and transmembrane domain of the RSV F protein; wherein the trimerization domain insertion site is the 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 RSVF protein is the F protein of type A or type B RSV virus, or a mutant with at least 80% sequence identity with it; And / or, the trimerization domain is selected from: Phage T4 minor fibrin fold and its variants, MTQ, heptapeptide ideal triple coiled helix and its variants, isoleucine zipper long chain and its variants, isoleucine zipper short chain and its variants, leucine zipper GCN4 and its variants, pulmonary surfactant-associated protein D trimer domain and its variants, collagen trimer domain and its variants, cartilage matrix protein-1 long chain trimer domain and its variants or cartilage matrix protein-1 short chain trimer domain and its variants; And / or, the heterologous transmembrane domain is selected from: the transmembrane domain of the HIV-1 virus gp160 protein and its variants, the transmembrane domain of the influenza virus HA protein and its variants, or the transmembrane domain of the novel coronavirus S protein and its variants.

3. The RSV F protein reconstructed polypeptide according to claim 1, characterized in that, The trimerization domain has an amino acid sequence selected from the following: SEQ ID NO: 5-14; And / or, the homologous transmembrane domain of the RSVF protein has an amino acid sequence as shown in SEQ ID NO: 60, 72, 73, 83; And / or, the heterologous transmembrane domain has an amino acid sequence selected from the following: SEQ ID NO: 61-63; And / or, the C-terminus of the transmembrane domain also includes an intracellular domain of its source protein; And / or, the linker has an amino acid sequence as shown in SEQ ID NO: 64 or SEQ ID NO:

65.

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

5. The RSVF protein reconstructed polypeptide according to claim 1, characterized in that, The RSV F protein reconstructed polypeptide further includes 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; 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 plasminogen activator tPA signal peptide.

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

8. A polynucleotide encoding an RSV F protein remodeling polypeptide as described in any one of claims 1-7.

9. The polynucleotide according to claim 8, characterized in that, The polynucleotide is a DNA molecule or an mRNA molecule.

10. A nucleic acid construct comprising a polynucleotide as described in claim 8 or 9, and at least one expression regulatory element operatively linked to said polynucleotide.

11. The nucleic acid construct according to claim 10, characterized in that, The nucleic acid construct comprises, or consists of, a DNA sequence as shown in one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75 or an RNA sequence corresponding to such sequences.

12. An expression vector comprising the nucleic acid construct as described in claim 10 or 11.

13. A host cell wherein it is transformed or transfected with the polynucleotide of claim 8 or 9, the nucleic acid construct of claim 10 or 11, or the expression vector of claim 12.

14. A recombinant respiratory syncytial virus (RSV) antigen, characterized in that, The RSV recombinant antigen comprises a multimer of the RSV F protein reconstructed polypeptide as described in any one of claims 1-7.

15. The RSV recombinant antigen according to claim 14, characterized in that, The RSV recombinant antigen comprises a trimer of the RSV F protein reconstructed polypeptide as described in any one of claims 1-7.

16. The use of the RSV F protein reconstructed polypeptide as described in any one of claims 1-7, the polynucleotide as described in claim 8 or 9, the nucleic acid construct as described in claim 10 or 11, the expression vector as described in claim 12, the host cell as described in claim 13, or the RSV recombinant antigen as described in claim 14 or 15 in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection.

17. The application according to claim 16, characterized in that, The vaccine also contains other antigenic components.

18. A vaccine or immunogenic composition comprising an RSV F protein reconstituted polypeptide as claimed in any one of claims 1-7, a polynucleotide as claimed in claim 8 or 9, a nucleic acid construct as claimed in claim 10 or 11, an expression vector as claimed in claim 12, a host cell as claimed in claim 13, or an RSV recombinant antigen as claimed in claim 14 or 15, and physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

19. The vaccine or immunogenic composition according to claim 18, which is a respiratory syncytial virus recombinant protein vaccine, comprising the RSV F protein reconstituted polypeptide as described in any one of claims 1-7 or the RSV recombinant antigen and adjuvant as described in claim 14 or 15.

20. The vaccine or immunogenic composition according to claim 19, characterized in that, The adjuvant is selected from one or more of the following adjuvants: aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, Matrix M2 adjuvant, and GLA-SE adjuvant.

21. The vaccine or immunogenic composition according to claim 18, wherein it is a respiratory syncytial virus DNA vaccine, the DNA vaccine comprising: (i) A eukaryotic expression vector comprising a DNA sequence encoding a reconstructed polypeptide of RSV F protein as described in any one of claims 1-7; and (ii) DNA vaccine adjuvants.

22. The vaccine or immunogenic composition according to claim 21, characterized in that, The DNA sequence encoding the RSV F protein reconstructed polypeptide as described in any one of claims 1-7 is a DNA sequence as shown in any one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 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 community-stimulating factor, human interleukin-12 and / or CpG oligonucleotide.

23. The vaccine or immunogenic composition according to claim 18, wherein it is a respiratory syncytial virus mRNA vaccine, said mRNA vaccine comprising: (I) The mRNA sequence encoding the RSVF protein reconstructed polypeptide as described in any one of claims 1-7; (II) mRNA vaccine adjuvants; and (III) Delivery carrier, wherein the delivery carrier is selected from one or more of lipid nanoparticles, cationic nanoemulsions, peptides, polymers, cationic peptide polymers, and cationic peptide lipid nanoparticles.

24. The vaccine or immunogenic composition according to claim 23, characterized in that, The mRNA sequence encoding the RSV F protein reconstructed polypeptide as described in any one of claims 1-7 is an mRNA sequence corresponding to the DNA sequence shown in any one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 75; And / or, the mRNA vaccine adjuvant is selected from: lipid nanoparticles, emulsions, Toll-like receptor agonists and / or CpG oligonucleotides.

25. The vaccine or immunogenic composition according to claim 18, which is a respiratory syncytial virus-viral vector vaccine, comprising: (1) Viral backbone vector; and (2) The DNA sequence encoding the RSVF protein reconstructed polypeptide as described in any one of claims 1-7, which is constructed into the viral backbone vector.

26. The vaccine or immunogenic composition according to claim 25, characterized in that, The DNA sequence encoding the RSV F protein reconstructed polypeptide as described in any one of claims 1-7 is a DNA sequence as shown in any one of SEQ ID NO: 43, 46, 49, 52, 54, 56, 57, 58, 59, 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.

27. The vaccine or immunogenic composition according to claim 18, which is a respiratory syncytial virus nanoparticle vaccine, comprising the RSV F protein reconstructed polypeptide and nanoparticle carrier as described in any one of claims 1-7.

28. The vaccine or immunogenic composition according to claim 27, characterized in that, The nanoparticle carrier is ferritin, and the RSVF protein reconstructed polypeptide as described in any one of claims 1-7 is covalently linked to ferritin and self-assembled into nanoparticles, so that the RSVF protein reconstructed polypeptide is present on the surface of the nanoparticles.

29. The vaccine or immunogenic composition according to any one of claims 18-28, characterized in that, The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, or parenteral formulation.

30. The vaccine or immunogenic composition according to claim 29, characterized in that, The nasal spray is selected from aerosols, sprays, and powders; And / or, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments; And / or, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

31. The method for preparing the RSV F protein reconstructed polypeptide according to any one of claims 1-4, characterized in that, The preparation method includes: The RSVF protein reconstructed polypeptide as described in any one of claims 1-4 is cloned and expressed by adding a nucleotide sequence encoding a signal peptide to the 5' end of a codon-optimized nucleotide sequence, a nucleotide sequence with a histidine tag and a stop codon to the 3' end, screening for correct recombinants, transfecting them into expression system cells for expression, collecting cell culture supernatant, and isolating the RSVF protein reconstructed polypeptide from it.

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