CN119161495B

CN119161495B - Respiratory syncytial virus reconstructed polypeptide and its application

Info

Publication number: CN119161495B
Application number: CN202411660083.7A
Authority: CN
Inventors: 蔡雨恒; 张文静; 张景明; 彭薇
Original assignee: Transsion Biomedicine Suzhou Co ltd
Current assignee: Transsion Biomedicine Suzhou Co ltd
Priority date: 2024-11-20
Filing date: 2024-11-20
Publication date: 2025-04-11
Anticipated expiration: 2044-11-20
Also published as: CN119161495A

Abstract

The present invention relates to a Respiratory Syncytial Virus (RSV) F protein reconstitution polypeptide, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell into which the above polynucleotide, nucleic acid construct or expression vector has been transformed or transfected, a stabilized multimer formed from the reconstitution polypeptide, an immunogenic composition comprising any of the foregoing, and the use thereof in the manufacture of a vaccine for the prevention and/or treatment of respiratory syncytial virus infection. The RSV F protein reconstitution polypeptide has good immunogenicity, can excite organisms to generate high-level neutralizing antibody titer, and has great significance for clinical treatment, prevention and control of respiratory syncytial virus.

Description

Respiratory syncytial virus reconstruction polypeptide and application thereof

Technical Field

The invention relates to the technical field of vaccines, in particular to a respiratory syncytial virus reconstruction polypeptide, a respiratory syncytial virus recombinant antigen formed by the respiratory syncytial virus reconstruction polypeptide, an immunogenic composition containing the respiratory syncytial virus recombinant antigen, a preparation method and application of the respiratory syncytial virus recombinant antigen.

Background

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 need for safe and effective RSV vaccines.

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

There has been a great deal of research on the conformational stability of RSV F proteins prior to fusion, but most of the research has focused on mutating amino acids at specific sites of the RSV F protein or introducing trimerization domains at the C-terminus of the F protein. In previous studies, the method of introducing a trimerization domain at the C-terminus of the F protein was to replace the transmembrane domain (TM) of the F protein with a trimerization domain. However, the effect of deletion of the transmembrane domain on vaccine protection after introduction of the trimerization domain has not been studied in depth.

Disclosure of Invention

The present invention provides a recombinant polypeptide based on the F protein before Respiratory Syncytial Virus (RSV) fusion, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell into which the above polynucleotide, nucleic acid construct or expression vector has been transformed or transfected, a stabilized multimer formed from the recombinant polypeptide, an immunogenic composition comprising any of the foregoing, and use thereof in the manufacture of a vaccine for the prevention and/or treatment of respiratory syncytial virus infection.

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides a Respiratory Syncytial Virus (RSV) reconstitution polypeptide, which sequentially comprises domains of an extracellular domain, a trimerization domain and a transmembrane domain of an RSV F protein, which are directly connected with each other or connected with each other through a linker, from the N-terminal to the C-terminal, wherein the trimerization domain insertion site is a corresponding site at the 513 st amino acid position of an 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.

As the above-mentioned RSV F protein reconstitution polypeptide, the RSV F protein may be an F0 protein derived from any RSV subtype or any RSV strain, and for example, may be a wild-type F protein derived from RSV strain A2 (Uniprot No. P03420), strain B1 (Uniprot No. O36634), strain 18537 (Uniprot No. P13843), strain TX-79233 (Uniprot No. R9TCY 6) or the like. Exemplary F0 protein sequences are shown in SEQ ID NOS.1-4.

The RSV F protein may be a mutant of F0 protein, and the mutant of F protein may be any (PreF) mutant that retains the pre-fusion conformation of F protein, for example, it may be a PreF mutant ："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". described in the following, and preferably the RSV F protein is an F protein mutant having at least 80% amino acid sequence identity with the corresponding wild-type F protein.

In a possible embodiment, the RSV F protein is an F protein of an RSV virus of type a or type B, or a mutant having at least 80% sequence identity thereto.

In a possible embodiment, the trimerization domain is selected from the group consisting of:

Phage T4 minor fibrin folder (T4 foldon) and variants thereof (e.g., having the sequence shown in SEQ ID NO: 5), MTQ (e.g., having the sequence shown in SEQ ID NO: 6), heptapeptide ideal triple-coiled coil (e.g., having the sequence shown in SEQ ID NO: 7), isoleucine Zipper (IZ) long chain (e.g., having the sequence shown in SEQ ID NO: 8), IZ short chain (e.g., having the sequence shown in SEQ ID NO: 9), leucine zipper GCN4 and variants thereof (e.g., having the sequence shown in SEQ ID NO: 10), lung surfactant related protein D trimerization domain (e.g., having the sequence shown in SEQ ID NO: 11), collagen trimerization domain (e.g., having the sequence shown in SEQ ID NO: 12), cartilage matrix protein-1 long chain trimerization domain (e.g., having the sequence shown in SEQ ID NO: 13), or cartilage matrix protein-1 short chain trimerization domain (e.g., having the sequence shown in SEQ ID NO: 14), or other trimerization domain.

In a possible embodiment, the transmembrane domain of the RSV F protein is the homologous transmembrane domain F/TM of the F protein (e.g., the sequences shown in SEQ ID NO:60, 72, 73), or is a heterologous transmembrane domain, such as the transmembrane domain gp160/TM of the HIV-1 virus gp160 protein (e.g., shown in SEQ ID NO: 61), the transmembrane domain HA/TM of the influenza virus HA protein (e.g., the sequences shown in SEQ ID NO: 62), the transmembrane domain S/TM of the novel coronavirus S protein (e.g., the sequences shown in SEQ ID NO: 63), or is another transmembrane domain.

Furthermore, in a possible embodiment, the C-terminal end of the transmembrane domain further comprises an intracellular domain of the protein from which it is derived. For example, when the transmembrane domain is a homologous transmembrane domain of an RSV F protein, the C-terminus of the homologous transmembrane domain further comprises an intracellular domain of the RSV F protein, and when the transmembrane domain is a heterologous transmembrane domain, the C-terminus of the heterologous transmembrane domain further comprises an intracellular domain of the heterologous protein from which it is derived.

Furthermore, in a possible embodiment, the linker has an amino acid sequence as shown in SEQ ID NO. 64 or SEQ ID NO. 65.

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

SEQ ID NO:24、27、30、33、35、37、38、39、40、74。

In a possible embodiment, the RSV F protein reconstitution polypeptide further comprises a signal peptide, which may be a homologous signal peptide or a heterologous signal peptide, optionally 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.

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

Preferably, the heterologous signal peptide has an amino acid sequence as shown in one of SEQ ID NOs 15 to 21;

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

In a second aspect, the invention provides a polynucleotide encoding a RSV F protein reconstitution polypeptide according to the first aspect described above.

In particular embodiments, the polynucleotide may be DNA or mRNA, preferably the polynucleotide is a nucleotide sequence optimized for human codons.

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

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

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

In a fifth aspect, the present invention provides a host cell transformed or transfected with a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above or an expression vector as described in the fourth aspect above;

alternatively, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell;

further alternatively, the mammalian cell is a 293T cell, a 293F cell, or a CHO cell;

further alternatively, the bacterial cell is an E.coli cell.

In a sixth aspect, the present invention provides a recombinant Respiratory Syncytial Virus (RSV) antigen comprising a multimer, preferably a trimer, of the RSV F protein reconstitution polypeptides described in the first aspect above.

In a seventh aspect, the present invention provides the use of a RSV F protein reconstitution polypeptide according to the first aspect, a polynucleotide according to the second aspect, a nucleic acid construct according to the third aspect, an expression vector according to the fourth aspect, a host cell according to the fifth aspect or a RSV recombinant antigen according to the sixth aspect for the manufacture of a vaccine for the prevention and/or treatment of respiratory syncytial virus infection.

In a possible embodiment, the vaccine wherein the RSV F protein reconstitution polypeptide according to the first aspect, the polynucleotide according to the second aspect, the nucleic acid construct according to the third aspect, the expression vector according to the fourth aspect, the host cell according to the fifth aspect or the RSV recombinant antigen according to the sixth aspect is the sole antigen component.

In other possible embodiments, the vaccine comprises other antigenic components in addition to the RSV F protein reconstitution 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 an eighth aspect, the invention provides a vaccine or immunogenic composition comprising an RSV F protein reconstitution polypeptide as described in the first aspect, a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, an expression vector as described in the fourth aspect, a host cell as described in the fifth aspect or an RSV recombinant antigen as described in the sixth aspect, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent.

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

Optionally, the adjuvant is one or more selected from aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, matrix M2 adjuvant, GLA-SE adjuvant.

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

(i) A eukaryotic expression vector comprising a DNA sequence encoding a recombinant polypeptide of the RSV F protein described in the first aspect above, and

(Ii) DNA vaccine adjuvants;

Optionally, the DNA sequence encoding the RSV F protein reconstitution polypeptide according to the first aspect is a DNA sequence as set forth in one of SEQ ID NOs 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

alternatively, the eukaryotic expression vector is selected from the group consisting of pGX0001, pVAX1, pCAGGS, and pcDNA series vectors;

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

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

(I) An mRNA sequence encoding the RSV F protein reconstitution polypeptide according to the first aspect above;

(II) mRNA vaccine adjuvant, and

(III) a delivery vehicle selected from one or more of lipid nanoparticles, cationic nanoemulsions, polypeptides, polymers, cationic polypeptide lipid nanoparticles, or combinations thereof.

Alternatively, the mRNA sequence encoding the recombinant polypeptide of RSV F protein described in the first aspect is an mRNA sequence corresponding to the DNA sequence as set forth in one of SEQ ID NOs 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

optionally, the mRNA vaccine adjuvant is selected from the group consisting of lipid nanoparticles, emulsions, toll-like receptor agonists and/or CpG oligonucleotides.

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

(1) Viral backbone vectors, and

(2) A DNA sequence encoding the RSV F protein reconstitution polypeptide according to the first aspect described above constructed into said viral backbone vector;

optionally, the viral backbone vector is selected from one or more of adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.

In other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus nanoparticle vaccine comprising an RSV F protein reconstitution polypeptide as described in the first aspect above and a nanoparticle carrier.

Optionally, the nanoparticle carrier is ferritin, and the RSV F protein reconstitution polypeptide according to the first aspect is covalently linked to ferritin and self-assembled into a nanoparticle, such that the RSV F protein reconstitution polypeptide is presented on the surface of the nanoparticle.

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

preferably, the nasal spray is selected from the group consisting of aerosols, sprays and powder sprays;

Preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, soft/hard capsules, film coatings and ointments;

Preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.

In a ninth aspect, the present invention provides a method for preparing the RSV F protein reconstitution polypeptide according to the first aspect, wherein the method comprises:

Cloning and expressing the nucleotide sequence encoding the signal peptide at the 5 'end and the nucleotide sequence encoding the histidine tag at the 3' end of the nucleotide sequence optimized by the codons encoding the recombinant polypeptide of the RSV F protein in the first aspect, screening the correct recombinants, transfecting cells of an expression system for expression, collecting cell culture supernatant, and separating the cell culture supernatant to obtain the recombinant polypeptide of the RSV F protein.

In one possible implementation of the above method, the expression system cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally the mammalian cell is a 293T cell, a 293F cell, or a CHO cell, optionally the bacterial cell is an E.coli cell.

In a tenth aspect, the present invention provides a method of preventing and/or treating respiratory syncytial virus infection comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a RSV F protein reconstitution polypeptide as defined in the first aspect, a polynucleotide as defined in the second aspect, a nucleic acid construct as defined in the third aspect, an expression vector as defined in the fourth aspect, a host cell as defined in the fifth aspect, a RSV recombinant antigen as defined in the sixth aspect and/or a vaccine or immunogenic composition as defined in the eighth aspect.

The "prophylactically and/or therapeutically effective amount" may vary depending on the subject to be administered, the organ of the subject, the symptoms, the administration method, etc., and may be determined by considering the type of dosage form, the administration method, the age and weight of the subject, the symptoms of the subject, etc., and the judgment of the veterinarian.

Advantageous effects

The invention designs the RSV F protein reconstruction polypeptide by introducing a trimerization domain into a specific site of the RSV F protein before fusion and connecting a homologous transmembrane domain or a heterologous transmembrane domain of the RSV F protein at the C end of the trimerization domain, and experiments prove that the RSV F protein reconstruction polypeptide has more stable fusion conformation and enhanced trimerization protein stability, thereby leading to stronger immunogenicity and higher immune response effect. The RSV F protein reconstitution polypeptide has good immunogenicity, can excite organisms to generate high-level neutralizing antibody titer, and has great significance for clinical treatment, prevention and control of respiratory syncytial virus.

In addition, the invention has proved through experiments that the combination of different trimerization domains and transmembrane domains of different sources can induce higher levels of neutralizing antibody titer, which indicates that the design mode is independent of specific RSV subtype or specific F protein or mutant thereof, and can realize higher neutralizing antibody titer level in a plurality of different mutants. Briefly, the RSV F protein reconstitution polypeptides of the invention have broad spectrum effectiveness.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 shows a schematic structure of the polypeptide described in example 1.

FIG. 2 shows a schematic diagram of the structure of mRNA described in example 2.

FIG. 3 shows representative ELISA detection results of mRNA-LNP encoding a pre-fusion F protein (PreF) mutant expressed in HEK293 cells as described in example 3.

FIG. 4 shows representative flow cytometric detection results of mRNA-LNP encoding a pre-fusion F protein (PreF) mutant described in example 3 expressed in HEK293 cells labeled with PreF-epitope antibodies.

FIG. 5 shows representative flow cytometric assay results of mRNA-LNP encoding a pre-fusion F protein (PreF) mutant described in example 3 expressed in HEK293 cells labeled with an F protein IV epitope antibody.

FIG. 6 shows the immunogenicity of mRNA-LNP in mice encoding the different PreF protein mutants containing the phage T4 minor fibrin folder (T4 foldon) trimerization domain described in example 5, wherein A-D show the neutralizing antibody titers of mRNA-LNP immunized mouse sera encoding the different PreF mutants PreF1, preF2, preF3, preF4, respectively, and the neutralizing antibody titers are shown as a histogram of NT50 Geometric Mean Titer (GMT) and the GMT values are marked on top of the histogram, with the error bars representing 95% CI (confidence interval).

FIG. 7 shows the immunogenicity of mRNA-LNP encoding the mutant of PreF protein containing MTQ or Isoleucine Zipper (IZ) short chain trimerization domain in mice described in example 5, and shows the neutralizing antibody titer of mRNA-LNP-immunized mouse serum encoding PreF1-IZ, preF1-IZ-F/TM _A、PreF1-MTQ、PreF1-MTQ-F/TM_A. Neutralizing antibody titers are shown as NT50 GMT histograms, with GMT values noted at the top of the histogram and error bars representing 95% ci.

FIG. 8 shows the immunogenicity of mRNA-LNP encoding the PreF mutant containing different transmembrane domains (TMs) described in example 5 in mice, and shows the neutralizing antibody titers of mRNA-LNP immunized mouse serum encoding TMs containing influenza HA or novel coronavirus S protein, HIV-1 gp160, RSV F protein, preF1-T4-HA/TM, preF1-T4-S/TM, preF1-T4-gp160/TM, preF1-T4-F/TM _A、PreF1-T4-F/TM_A525-550 and PreF1-T4, preF1-F/TM _A as controls. Neutralizing antibody titers are shown as NT50 GMT histograms, with GMT values noted at the top of the histogram and error bars representing 95% ci.

Detailed Description

For a better understanding of the present invention, definitions and explanations of the relevant terms are provided below.

The term "polypeptide" refers to any chain of amino acids, whether in length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptides" are applicable to amino acid polymers, including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as artificial chemical mimics in which one or more amino acid residues are non-natural amino acids, e.g., corresponding naturally occurring amino acids. "residue" refers to an amino acid or amino acid mimetic that is incorporated into a polypeptide by an amide bond or amide bond mimetic. The polypeptide has an amino-terminal (N-terminal) end and a carboxy-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.

The term "F protein" or "F polypeptide" refers to a protein or polypeptide having the entire or partial amino acid sequence of an RSV F protein.

The RSV F protein, i.e., fusion protein or Fusion protein, is an RSV envelope glycoprotein capable of promoting Fusion of the RSV viral membrane and host cell membrane, and plays a vital role in respiratory syncytial virus infection process. In nature, the RSV F protein was initially synthesized as a single polypeptide precursor of about 574 amino acids in length, designated F0. F0 includes an N-terminal signal peptide that directs localization to the endoplasmic reticulum, wherein the signal peptide (approximately the first 25 residues of F0) is proteolytically cleaved. The remaining F0 residues are trimerized by oligomerization and proteolytic processing by cellular proteases at two conserved consensus furin cleavage sequences (approximately at amino acid residues 109 and 136 of F0; e.g., RARR ₁₀₉ (amino acid residues 106-109 of F0) and RKRR ₁₃₆ (amino acid residues 133-136 of F0) to produce two disulfide-linked fragments F1 and F2., the smaller F2 of which is derived from the N-terminal portion of the F0 precursor and comprises approximately amino acid residues 26-109 of F0. The larger F1 of which comprises the C-terminal portion of the F0 precursor (approximately amino acid residues 137-574 of F0), the extracellular domain comprising the extracellular/luminal region (approximately amino acid residues 137-529), the transmembrane domain (approximately amino acid residues 551-550) and the cytoplasmic tail (approximately amino acid residues 574) of the C-terminal RSV F protein is the extracellular domain of RSV F2 comprising F2 and F1.

RSV F protein exhibits significant sequence conservation in the RSV subtype. For example, in the F0 precursor molecule, RSV subtypes a and B have 90% sequence identity, and RSV subtypes a and B each have 81% sequence identity to bovine RSV F protein. Within the RSV subtypes, F0 sequence identity is even higher, e.g., RSV F0 precursor proteins have about 98% sequence identity within each of the RSV subtypes a, B and Niu Ya. Almost all identified RSV F0 precursor proteins are about 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, one of ordinary skill in the art can readily compare amino acid positions between different natural RSV F sequences to identify corresponding RSV F amino acid positions between different RSV strains and subtypes. Thus, conservation of RSV F protein sequences across strains and subtypes allows the use of reference RSV F sequences to compare amino acids at specific positions in the RSV F protein. Unless the context indicates otherwise, numbering of amino acid substitutions disclosed herein is made with reference to the RSV F0 polypeptide sequences shown in SEQ ID NOS 1-4.

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

"Domain" or "domain" of a polypeptide or protein refers to a defined element of a structure within a polypeptide or protein, e.g., a "trimerization domain" refers to an amino acid sequence within a polypeptide that facilitates assembly of the polypeptide into a trimer. For example, trimerization domains promote assembly into trimers via association with other trimerization domains (of other polypeptides having the same or different amino acid sequences); "transmembrane domain" refers to an amino acid sequence capable of intercalating into a lipid bilayer (e.g., of a cell or virus-like particle) or anchoring an antigen to a membrane. The term is also used to refer to polynucleotides encoding such peptides or polypeptides. The domains in the RSV F protein reconstitution polypeptide of the invention can be directly connected, or can be connected through natural or artificial connectors, linkers and Spacer sequences (spacers).

The term "linker" or "Spacer" is a natural or artificial bifunctional molecule that can be used to link two molecules into one continuous molecule. Non-limiting examples of peptide linkers include fragments of any length between the glycine-serine peptide linker and amino acids 514-524 of RSV F protein. Unless the context indicates otherwise, reference to "linking" a first polypeptide to a second polypeptide, or "linking" two polypeptides together, or a first polypeptide having "linkage" with a second polypeptide, refers to covalent linkage through a peptide bond (e.g., through a peptide linker) such that the first and second polypeptides form a continuous polypeptide chain. If a peptide linker is involved, the covalent attachment of the first and second polypeptides may be to the N-and C-terminus of the peptide linker. The term "mutant" 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 as compared to the native or reference sequence. Typically, the variant has at least 50% homology to a native or reference sequence. In some embodiments, the variant shares at least 80% or at least 90% homology with the native or reference sequence.

The term "homology" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Polymeric molecules (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 alignment of matching residues are referred to as homology. Homology is a qualitative term describing the relationship between molecules and may be based on quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence matching between two compared sequences. In some embodiments, polymeric molecules are considered "homologous" to each other 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 "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides encoded by the two 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. In some embodiments, the homologous polynucleotide sequence is characterized by the ability to encode an stretch of 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 of at least 4 to 5 uniquely specified amino acids. Two proteins are considered homologous in sequence if they are at least 50%, 60%, 70%, 80% or 90% identical for at least one stretch of at least 20 amino acids. The terms "polynucleotide" and "nucleic acid sequence" refer to polymeric forms of nucleotides that are at least 10 bases in length. The nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form of any nucleotide. The 5 'and 3' directions of the nucleic acid are defined by referring to the connectivity of the respective nucleotide units, and are assigned according to the carbon positions of deoxyribose (or ribose) sugar rings. The information (coding) content of the polynucleotide sequence is read in the 5 'to 3' direction.

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

In some embodiments, the nucleotide vaccine of the present 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 the RNA comprises at least one chemical modification.

The terms "chemically modified" and "chemically modified" refer to modification of at least one of its position, pattern, percentage or population with respect to adenosine (a), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides. In general, these terms do not refer to ribonucleotide modification of the cap portion of the naturally occurring 5' terminal mRNA. Modifications of polynucleotides include, but are not limited to, those described herein, and include (but are not limited to) those modifications comprising chemical modifications. A polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) may comprise naturally occurring, non-naturally occurring modifications or a polynucleotide may comprise a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may include any suitable modification to, for example, a sugar, nucleobase, or internucleoside linkage (e.g., to a linked phosphate, to a phosphodiester linkage, or to a phosphodiester backbone).

The term "heterologous" in reference to a nucleic acid, polypeptide, or other cellular component indicates the presence of a component that is not normally found in nature and/or that is derived from a different source or species.

An "antigen" refers to a compound, composition, or substance capable of stimulating antibody production and/or a T cell response in an animal, including a composition that is injected, adsorbed, or otherwise introduced into an animal. 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. "dominant antigenic epitope" or "dominant epitope" refers to those epitopes to which a functionally significant host immune response (e.g., an antibody response or a T cell response) is generated. Thus, with respect to a protective immune response against a pathogen, a dominant antigenic epitope refers to those antigenic moieties that when recognized by the host immune system produce protection against disease caused by the pathogen. The term "T cell epitope" refers to an epitope that is specifically bound by T cells (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).

"Vaccine" refers to a formulation of an immunogenic substance capable of stimulating an immune response that is administered for the prevention, amelioration, or treatment of infectious diseases or other types of diseases. The immunogenic material may include attenuated or killed microorganisms (e.g., bacteria or viruses), or antigenic proteins, peptides or DNA derived therefrom. The vaccine may include a disclosed immunogen (e.g., recombinant RSV F ectodomain trimer or nucleic acid molecule encoding the same), a virus, a cell, or one or more cellular components. Vaccines can elicit both prophylactic (prophylactic or protective) and therapeutic responses. The method of administration varies depending on the vaccine but may include vaccination, ingestion, inhalation or other forms of administration. The vaccine may be administered with an adjuvant to boost 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 as compared to a control.

The "carrier" or "excipient" refers to pharmaceutically acceptable auxiliary materials, refers to excipients and additives used in the production of medicines and formulation prescriptions, and is a substance which has been reasonably evaluated in terms of safety except for active ingredients and is contained in pharmaceutical preparations. The pharmaceutically acceptable auxiliary materials not only form, serve as carriers and improve stability, but also have important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which can influence the quality, safety and effectiveness of the medicine. Depending on the application and use, the pharmaceutical excipients may be divided into solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binders, integration agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarders, and the like.

A "recombinant" is a molecule having a sequence that does not occur in nature, e.g., comprising one or more nucleic acid substitutions, deletions or insertions, and/or having a sequence prepared by artificial combination of two otherwise separated sequence segments. Such artificial combination may be accomplished by chemical synthesis or more commonly by manipulation of isolated nucleic acid segments by hand (e.g., by genetic engineering techniques). Recombinant proteins are proteins having a non-naturally occurring sequence or having a sequence prepared by 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.

An "expression control element" refers to a nucleic acid sequence that modulates the expression of a heterologous nucleic acid sequence to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and (where appropriate) translation of the nucleic acid sequence. Thus, expression control sequences may include appropriate promoters, enhancers, transcription terminators, start codons (ATGs) prior to the protein encoding gene, splicing signals for introns (maintaining the correct reading frame of the gene to allow for correct translation of the mRNA), and stop codons. The term "regulatory element" is intended to include at least the components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The expression control element may include a promoter.

A "promoter" is a minimal sequence sufficient to direct transcription. Including T7 promoters, and also include those promoter elements sufficient to allow promoter-dependent gene expression to be controllable with respect to cell type specificity, tissue specificity, or to be inducible by external signals or agents, such elements may be located 5 'or 3' of the gene. Including constitutive and inducible promoters (see, e.g., bitter et al Methodsin Enzymology 153:153:516-544,1987). For example, when cloning in bacterial systems, inducible promoters such as pL, plac, ptrp, ptac of phage lambda (ptrp-lac hybrid promoter) and the like can be used. In one embodiment, when cloned in a mammalian cell system, promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., retrovirus long terminal repeat; adenovirus late promoter; vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of a nucleic acid sequence.

"5'UTR" refers to the region of mRNA that does not encode a polypeptide immediately upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome). "3'UTR" refers to the region of mRNA that does not encode a polypeptide immediately downstream (i.e., 3') of a stop codon (i.e., the codon of an mRNA transcript that signals translation termination).

"Coding sequence CDS" is a continuous stretch of RNA starting with a start codon (e.g., methionine (AUG)) and ending with a stop codon (e.g., UAA, UAG, or UGA) and encoding a polypeptide.

A "polyA tail" or "polyadenylation tail" is located in the mRNA downstream of the 3' UTR and contains multiple consecutive adenosine monophosphates. The polyA tail may contain 10 to 300 adenosine monophosphates. For example, the polyA tail may 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, nucleotides in the polyA tail that contain non-adenosines segment the contiguous adenosine monophosphate. In related biological contexts (e.g., in cells, in vivo), the poly (a) tail serves to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and to facilitate transcription termination and/or mRNA export and translation from the nucleus.

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

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.

Example 1 structural design of the reconstitution polypeptide

In this example, the following recombinant polypeptides of the invention and their control polypeptides were each designed.

The reconstructed polypeptide of the invention:

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), and PreF1-T4-F/TM _A525-550 (whose sequence is shown in SEQ ID NO: 74).

Control polypeptide:

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).

The sequence of each polypeptide is the sequence without the endogenous signal peptide, the endogenous signal peptide sequences used by the polypeptide are shown as SEQ ID NO. 66 or 67, wherein, the sequence of the polypeptide is shown as SEQ ID NO. 22, the sequence of the polypeptide is shown as SEQ ID NO. 23, the sequence of the polypeptide is shown as SEQ ID NO. 4, the sequence of the polypeptide is shown as SEQ ID NO. _A, the sequence of the polypeptide is shown as SEQ ID NO. 24, the sequence of the polypeptide is shown as SEQ ID NO. 1-T4-F/TM _A, 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), and, PreF3-T4 (the sequence of which is shown in SEQ ID NO: 29), preF3-T4-F/TM _A (the sequence of which is shown in SEQ ID NO: 30), preF1-MTQ (the sequence of which is shown in SEQ ID NO: 34), preF1-MTQ-F/TM _A (the sequence of which 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), The endogenous signal peptide sequence of PreF1-T4-F/TM _A525-550 (its sequence is shown as SEQ ID NO: 74) is shown as SEQ ID NO:66, and the endogenous signal peptide sequences of the remaining polypeptides are shown as SEQ ID NO: 67.

The schematic structure of each polypeptide is shown in figure 1.

EXAMPLE 2 preparation of mRNA for reconstituted polypeptide and packaging of Lipid Nanoparticles (LNP)

A pUC57 plasmid (construction service provided by Nanjing St Biotechnology Co., ltd.) containing the T7 promoter (SEQ ID NO: 68), 5'UTR (SEQ ID NO: 69), the coding sequence (CDS) of each reconstructed polypeptide, 3' UTR (SEQ ID NO: 70), polyadenylation tail (SEQ ID NO: 71), restriction enzyme site (sequence GAAGAGC) was constructed, which was subjected to enzyme-tangential linearization and purification to obtain a high-quality linearized plasmid as an in vitro transcription template. The linearized plasmid template is subjected to in vitro transcription under T7 RNA polymerase, 3' -OMe-GAG cap analogues, 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 resulting mRNA was analyzed by spectrophotometry (NanoDrop One, thermo Scientific) and capillary electrophoresis (Agilent 5200). The capping rate of mRNA and poly A tail were analyzed by liquid chromatography combined with mass spectrometry (LC-MS). The mRNA solution was also analyzed for pH, residual DNA, protein, and double stranded RNA impurities. FIG. 2 shows a schematic diagram of mRNA composition.

MRNA was encapsulated using LNP formulated from compound 5 from proprietary CN118084714B, DSPC, cholesterol, and DMG-PEG 2000. mRNA concentration, encapsulation efficiency, mRNA integrity, LNP particle size and Zeta potential of the mRNA-LNP samples were analyzed using a Quant-iTTM RiboGreenTM RNA ASSAY KIT (Invitrogen) -microplate reader (Varioskan LUX, thermo Scientific), capillary electrophoresis (Agilent 5200), light scattering (Zetasizer Ultra). The pH, endotoxin and bioburden of the mRNA-LNP samples were also analyzed, ensuring suitability for preclinical studies.

Using the procedure described above, mRNA-LNP for each reconstituted polypeptide was prepared as an mRNA vaccine.

Example 3 expression of mRNA-LNP encoding the recombinant polypeptide in HEK293 cells

MRNA-LNP encoding the reconstituted polypeptide was tested for expression in HEK293 cells using flow cytometry or enzyme-linked immunosorbent assay (ELISA). HEK293 cells are paved in a culture dish, when the cells grow to 80% coverage, mRNA-LNP is added, after transfection for 24 hours, the cells are collected, F protein on the cell surface is respectively marked by fluorescent antibodies for recognizing F protein IV epitope and PreF protein phi epitope (blank cells which are not transfected with mRNA are used as negative control), the cells are washed after incubation, and then the fluorescence intensity is detected by a flow cytometer. HEK293 cells are paved in a culture dish, when the cells grow to 80% and cover, mRNA-LNP is added, after 48 hours of transfection, culture supernatant is collected, sediment is removed, ultrafiltration tube with molecular weight of 10kDa is used for concentration of 15 times, then ELISA kit is used for detecting concentration of Pre F protein in supernatant concentrate, capture monoclonal antibody which specifically recognizes RSV PreF epitope and detection monoclonal antibody-horseradish peroxidase (HRP) which recognizes II/III epitope are used for detecting the content of Pre F based on a double antibody sandwich method.

FIG. 3 shows representative ELISA detection results of recombinant polypeptides encoding transmembrane-free domains, cells encoding mRNA-LNP of the recombinant polypeptides PreF1-T4, preF2-T4, preF3-T4, preF4-T4, preF1-IZ, and PreF1-MTQ were transfected, and the cell culture supernatants expressed the PreF protein. FIG. 4 shows the results of representative flow cytometry detection of recombinant polypeptides containing a transmembrane domain labeled with PreF-epitope antibodies, showing that the cell transfection of mRNA-LNP encoding 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 recombinant polypeptides resulted in a cell PreF protein positive rate of above 98%. FIG. 5 shows the results of representative flow cytometry detection of recombinant polypeptides containing the transmembrane domain labeled with F protein IV epitope antibodies, showing that the cells were transfected with mRNA-LNP encoding the PreF1-T4-F/TM _A525-550、PreF4-F/TM_B、PreF4-T4-F/TM_B, preF1-T4-gp160/TM, and PreF1-T4-S/TM recombinant polypeptides with a cell PreF protein positive rate between 17.4-99.3%. Wherein the cell F protein positive rate and the Mean Fluorescence Intensity (MFI) of the PreF1-T4-gp160/TM and PreF1-T4-S/TM reconstitution polypeptides are significantly better than those of the negative control group not transfected with mRNA-LNP, although lower than those of other reconstitution polypeptides. The positive rate of protein F in the cells of the negative control group, which were not transfected with mRNA-LNP, was 0.12%. The MFI of the PreF1-T4-gp160/TM and the PreF1-T4-S/TM reconstitution polypeptide was about 2 times that of the negative control group.

In this example, only the cell surface F protein was detected in the flow cytometry test, and only the protein secreted outside the cell was detected in the ELISA test, so that the detection value did not represent the total protein expression, and it was only confirmed that all mRNA-LNP was expressed to produce the F protein.

Taken together, the above results demonstrate that the recombinant polypeptides of the invention are expressed in cells.

Example 4 immunization and sample collection of Experimental animals

BALB/c females at 6-8 weeks of age were randomly grouped, 6 per group, each round of experiment contained a blank control in addition to the experimental group. On day 0, the mRNA-LNP vaccine was injected into the mice by intramuscular injection outside the right hind leg of the mice at a dose of 5. Mu.g/dose and an injection volume of 50. Mu.L/dose. The blank group was injected with only the same volume of physiological saline. On day 14, booster immunizations were performed in the same manner, at the same doses. The weight of the mice is monitored during the period, so that abnormal conditions are avoided. On day 28, whole blood was collected from the eyes, and serum was isolated.

Example 5 plaque reduction neutralization assay (FRNT) detection of neutralizing antibody titres in serum of immunized mice

Serum neutralizing antibody titer NT50 was measured using a foci reduction neutralization assay (FRNT). The F protein sequence of the vaccine is derived from neutralizing antibody detected by RSV-A2-GFP virus of type A strain and from neutralizing antibody detected by RSV-B1-GFP virus of type B strain.

HEP-2 cells were plated into 96-well cell culture plates and when the cells were grown to 95% coverage, the subsequent experiments were started. Serum complement inactivation-serum complement was inactivated for 30min in a 56 ℃ water bath prior to serum use. Serum was diluted with dilution (DMEM medium+10% fbs), starting at 40-fold dilution, then at 3-fold ratio for a total of 8 dilution gradients. Virus dilution the F protein sequence of the vaccine is derived from the neutralizing antibody detected by the RSV-A2-GFP virus of the A-type strain (PreF 1, preF2, preF3 related reconstitution polypeptide) and from the neutralizing antibody detected by the RSV-B1-GFP virus of the B-type strain (PreF 4 related reconstitution polypeptide). The virus stock was diluted to 10 ⁴ pfu/mL. Virus neutralization, namely, respectively taking 220 mu L of serum diluent and virus diluent, uniformly mixing, and placing the mixture in a 37 ℃ incubator for incubation for 1h. Serum-virus mixture was added to HEP-2 cell 96 well plates, cell culture supernatants were removed, 200. Mu.L of neutralized serum-virus mixture was added to each well, two duplicate wells were made for each serum dilution, and cell culture plates were placed in a 37℃incubator for 48h. And (3) fluorescent spot reading, namely removing the culture medium, washing the cell surface for 2 times by using PBS, taking the liquid, reading focus spot counting results by using an enzyme-linked spot analyzer, and finally calculating the titer of the RSV neutralizing antibody by combining the titer of the virus and the dilution of serum.

The results and analysis were as follows:

1) Neutralizing antibody titre induced by mRNA vaccines based on different RSV F proteins for the reconstituted polypeptides of the invention

The levels of neutralizing antibody titers induced by mRNA vaccines based on the recombinant polypeptides of the present invention of PreF1, preF2, preF3, preF4 are shown in fig. 6, a-D, respectively, which shows that each PreF antigen containing both the trimerization domain T4 foldon and the transmembrane domain F/TM (i.e., the recombinant polypeptide of the present invention) induces neutralizing antibodies on average higher than the antigen containing either T4 alone or F/TM alone (i.e., the control polypeptide). The Geometric Mean Titers (GMT) of neutralizing antibodies induced by PreF1-T4-F/TM _A were 2.88 and 2.70 times that of PreF1-F/TM _A and PreF1-T4, respectively. The neutralizing antibodies GMT induced by PreF2-T4-F/TM _A are 1.77 and 1.14 times that of PreF2-F/TM _A and PreF2-T4, respectively. The neutralizing antibodies GMT induced by PreF3-T4-F/TM _A are 6.91 and 1.71 times that of PreF3-F/TM _A and PreF3-T4, respectively. The neutralizing antibodies GMT induced by PreF4-T4-F/TM _B are 2.04 and 1.43 times that of PreF4-F/TM _B and PreF4-T4, respectively. This demonstrates that the ability of the reconstituted polypeptides of the invention to induce neutralizing antibodies is significantly better than the control polypeptides. This demonstrates that the immunogenicity can be significantly improved by linking the transmembrane domain to the C-terminus of the trimerisation domain.

2) Neutralizing antibody titre induced by mRNA vaccines based on different trimerization domains for the reconstituted polypeptides of the invention

FIG. 7 shows the level of neutralizing antibody titer induced by mRNA vaccines based on the recombinant polypeptides of the present invention with IZ or MTQ, which shows that mRNA vaccines containing the recombinant polypeptides of the present invention with trimerization domains IZ or MTQ each induced a higher level of neutralizing antibody titer, which suggests that the recombinant polypeptides of the present invention with different trimerization domains IZ or MTQ each induced a higher neutralizing antibody titer, and that they were both higher than the control mRNA vaccine containing only trimerization domains but no transmembrane domains, the neutralizing antibody GMT induced by PreF1-IZ-F/TM _A was 6.77 times that of PreF1-IZ, and the neutralizing antibody GMT induced by PreF1-MTQ-F/TM _A was 1.66 times that of PreF1-MTQ, which suggests that by linking the transmembrane domains at the C-terminus of the trimerization domains, the immunogenicity was significantly improved.

3) Neutralizing antibody titre induced by mRNA vaccines based on different transmembrane domains for the reconstituted polypeptides of the invention

FIG. 8 shows neutralizing antibody titers induced by mRNA vaccines employing the recombinant polypeptides of the present invention with different transmembrane domains (homologous or heterologous), which shows that mRNA vaccines containing the recombinant polypeptides of the present invention with different transmembrane domains each induce higher levels of neutralizing antibody titers, which are higher than corresponding control mRNA vaccines containing either the trimerization domain alone or the F protein self-transmembrane domain F/TM alone. The neutralizing antibodies GMT induced by PreF1-T4-HA/TM, preF1-T4-S/TM, preF1-T4-gp160/TM and PreF1-T4-F/TM _A、PreF1-T4-F/TM_A525-550.05, 2.43, 1.71, 2.70 and 3.66 times that of PreF1-T4, respectively, 5.39, 2.60, 1.82, 2.88 and 3.91 times that of PreF1-F/TM _A, respectively. This demonstrates that by linking the homologous or heterologous transmembrane domains to the C-terminus of the trimerisation domain, the immunogenicity can be significantly increased.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A Respiratory Syncytial Virus (RSV) F protein reconstruction polypeptide is characterized in that the RSV F protein reconstruction polypeptide sequentially comprises domains which are directly connected with each other or connected with each other through a linker from the N end to the C end, wherein the trimerization domain is selected from a bacteriophage T4 minor fibrin folder, an MTQ and an isoleucine zipper short chain, the insertion site of the trimerization domain is a corresponding site with reference to the 513 th position of the amino acid of the RSV F protein precursor polypeptide shown as 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 reconstitution polypeptide of claim 1, wherein the RSV F protein is F protein of an a-type or B-type 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 recombinant polypeptide of RSV F protein of claim 1, wherein the amino acid sequence of the trimerization domain is selected from the group consisting of SEQ ID NOs 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-terminal of the transmembrane domain further comprises an intracellular domain of a protein from which it is derived;

And/or the amino acid sequence of the linker is shown as SEQ ID NO. 64 or SEQ ID NO. 65.

4. The RSV F protein reconstitution polypeptide of claim 1, wherein the amino acid sequence of the RSV F protein reconstitution polypeptide is selected from the group consisting of:

SEQ ID NO:24、27、30、33、35、37、38、39、40、74。

5. The RSV F protein reconstitution polypeptide of claim 1, wherein the RSV F protein reconstitution polypeptide further comprises a signal peptide.

6. The RSV F protein reconstitution polypeptide of claim 5, wherein the signal peptide is a homologous signal peptide or a heterologous signal peptide.

7. The RSV F protein reconstitution polypeptide of claim 6, wherein 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.

8. The recombinant polypeptide of RSV F protein of claim 6 wherein the amino acid sequence of the homologous signal peptide is selected from the group consisting of SEQ ID NOs 66-67;

And/or the amino acid sequence of the heterologous signal peptide is selected from the group consisting of SEQ ID NOS 15-21.

9. A polynucleotide encoding the RSV F protein reconstitution polypeptide of any one of claims 1-8.

10. The polynucleotide of claim 9, wherein the polynucleotide is a DNA molecule or an mRNA molecule.

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

12. The nucleic acid construct of claim 11, wherein the nucleic acid construct comprises or consists of a DNA sequence as set forth in one of SEQ ID NOs 43, 46, 49, 52, 54, 56, 57, 58, 59, 75 or an RNA sequence corresponding to such sequences.

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

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

15. A recombinant Respiratory Syncytial Virus (RSV) antigen comprising the multimer of the RSV F protein reconstitution polypeptide of any one of claims 1-8.

16. The RSV recombinant antigen of claim 15, wherein the RSV recombinant antigen comprises a trimer of RSV F protein reconstitution polypeptides of any one of claims 1-8.

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

18. The use according to claim 17, wherein the vaccine further comprises other antigenic components.

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

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

21. The vaccine or immunogenic composition according to claim 20, wherein the adjuvant is one or more selected from the group consisting of aluminium adjuvant, MF59 adjuvant, AS01 adjuvant, matrix M2 adjuvant, GLA-SE adjuvant.

22. The vaccine or immunogenic composition of 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 reconstitution polypeptide of any of claims 1-8, and

(Ii) DNA vaccine adjuvants.

23. The vaccine or immunogenic composition according to claim 22, wherein the DNA sequence encoding the RSV F protein reconstitution polypeptide according to any one of claims 1-7 is a DNA sequence as set forth in one of SEQ ID NOs 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 colony stimulating factor, human interleukin-12 and/or CpG oligonucleotide.

24. The vaccine or immunogenic composition of claim 19, which is a respiratory syncytial virus mRNA vaccine comprising:

(I) An mRNA sequence encoding the RSV F protein reconstitution polypeptide of any one of claims 1-8;

(II) mRNA vaccine adjuvant, and

25. The vaccine or immunogenic composition according to claim 24, wherein the mRNA sequence encoding the RSV F protein reconstitution polypeptide according to any one of claims 1-8 is an mRNA sequence corresponding to the DNA sequence as set forth in one of SEQ ID NOs 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

And/or the mRNA vaccine adjuvant is selected from the group consisting of lipid nanoparticles, emulsions, toll-like receptor agonists, and/or CpG oligonucleotides.

26. The vaccine or immunogenic composition of claim 19, which is a respiratory syncytial virus-viral vector vaccine comprising:

(1) Viral backbone vectors, and

(2) A DNA sequence encoding the RSV F protein reconstitution polypeptide of any one of claims 1-8 constructed into said viral backbone vector.

27. The vaccine or immunogenic composition according to claim 26, wherein the DNA sequence encoding the RSV F protein reconstitution polypeptide according to any one of claims 1-8 is a DNA sequence as set forth in one of SEQ ID NOs 43, 46, 49, 52, 54, 56, 57, 58, 59, 75;

And/or the virus skeleton vector is selected from one or more of adenovirus vector, poxvirus vector, influenza virus vector and adeno-associated virus vector.

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

29. The vaccine or immunogenic composition of claim 28, wherein the nanoparticle carrier is ferritin, and the RSV F protein reconstitution polypeptide of any one of claims 1-8 is covalently linked to ferritin and self-assembled into a nanoparticle such that the RSV F protein reconstitution polypeptide is presented on the surface of the nanoparticle.

30. The vaccine or immunogenic composition according to any one of claims 19-29, wherein 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, wherein the nasal spray is selected from the group consisting of aerosols, sprays and powder sprays;

And/or the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, soft/hard capsules, film coatings and ointments;

And/or the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.

32. The method of any one of claims 1-4, wherein the method comprises:

Cloning and expressing the nucleotide sequence encoding the recombinant protein F polypeptide of RSV according to any one of claims 1-4, adding the nucleotide sequence encoding signal peptide to the 5 'end of the nucleotide sequence optimized by the codon, adding the nucleotide sequence tagged with histidine and the stop codon to the 3' end, screening the correct recombinant, then transfecting the cell of the expression system for expression, collecting the cell culture supernatant, and separating the cell culture supernatant to obtain the recombinant polypeptide of RSV F protein F.