Anti-respiratory syncytial virus antigen and use thereof

Abstract

The present application relates to a respiratory syncytial virus antigen, comprising, but not limited to, a protein or 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 polynucleotide, the nucleic acid construct or the expression vector, a stabilized multimer formed from the antigen, an immunogenic composition comprising any one of the foregoing, and the use thereof in the preparation of a vaccine / drug for preventing and / or treating respiratory syncytial virus infection.

Description

Antigens against respiratory syncytial virus infection and their applications

[0001] Cross-references

[0002] This application claims priority to Chinese patent application filed on November 27, 2024, application number 202411718386.X, entitled "Antigens against Respiratory Syncytial Virus Infection and Their Applications", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of immunology, specifically to a respiratory syncytial virus antigen, an immunogenic composition comprising the antigen, and the use thereof. Background Technology

[0004] Respiratory syncytial virus (RSV) is an enveloped, non-segmental, negative-sense RNA virus belonging to the genus Pneumovirus in the family Paramyxoviridae. It is the most common cause of bronchiolitis and pneumonia in children during their first year of life. RSV also causes recurrent infections, including severe lower respiratory tract illnesses, which can occur at any age, but are particularly prevalent in older adults or those with compromised heart, lungs, or immune systems. Passive immunization is currently used to prevent severe illness caused by RSV infection, especially in premature infants, those with bronchopulmonary dysplasia, or those with congenital heart disease.

[0005] Currently, the RSV fusion protein (F protein) is considered an ideal target for RSV vaccines. Immunological responses have been enhanced by mutating specific amino acid sites on the F protein and introducing trimer domains to maintain its pre-fusion conformation. However, the selection of sites for introducing trimer domains has not been thoroughly investigated. Summary of the Invention

[0006] This application provides an antigen (including but not limited to a protein or polypeptide) for resisting respiratory syncytial virus (RSV) infection based on the pre-fusion F protein of 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 aforementioned polynucleotide, nucleic acid construct or expression vector, a stabilized multimer formed by the F protein-based RSV antigen, an immunogenic composition comprising any of the foregoing, and its use in the preparation of a vaccine / medication for the prevention and / or treatment of respiratory syncytial virus infection.

[0007] Specifically, this application provides the following technical solutions:

[0008] In a first aspect, a respiratory syncytial virus (RSV) antigen based on the F protein, the antigen comprising the following domains sequentially linked from the N-terminus to the C-terminus: an extracellular domain, a trimerization domain, and a transmembrane domain of the RSV F protein; wherein the trimerization domain is inserted at any of amino acid positions 514 to 524 of the wild-type RSV F protein sequence as shown in SEQ ID Nos. 28 to 31, or at a corresponding position thereof.

[0009] For the aforementioned RSV antigen based on the F protein, the RSV F protein can be a wild-type (F0) protein derived from any RSV subtype or strain, such as 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 Nos. 28 to 31.

[0010] 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 RSV F protein is an F protein mutant with at least 80% amino acid sequence identity to the corresponding wild-type F protein, or an F protein mutant with at least 85% amino acid sequence identity to the corresponding wild-type F protein, or an F protein mutant with at least 90% amino acid sequence identity to the corresponding wild-type F protein, or an F protein mutant with at least 95% amino acid sequence identity to the corresponding wild-type F protein, or an F protein mutant with at least 99% amino acid sequence identity to the corresponding wild-type F protein.

[0011] In a feasible implementation, the RSV F protein is the F protein of type A or type B RSV virus, or a mutant with at least 80% amino acid sequence identity, or a mutant with at least 85% amino acid sequence identity, or a mutant with at least 90% amino acid sequence identity, or a mutant with at least 95% amino acid sequence identity, or a mutant with at least 99% amino acid sequence identity.

[0012] In a feasible implementation, the trimerization domain is selected from:

[0013] Phage T4 minor fibrin foldon and its variants (SEQ ID No. 1), MTQ (SEQ ID No. 2), heptapeptide ideal triple coiled helix (SEQ ID No. 165), isoleucine zipper (IZ) long chain (SEQ ID No. 166), IZ short chain (SEQ ID No. 3), leucine zipper GCN4 and its variants (SEQ ID No. 167), pulmonary surfactant-associated protein D trimer domain (SEQ ID No. 168), collagen trimer domain (SEQ ID No. 169), chondromalin-1 long chain trimer domain (SEQ ID No. 170) or chondromalin-1 short chain trimer domain (SEQ ID No. 171) or other trimer domains.

[0014] In feasible implementations, the transmembrane domain is the RSVF protein homologous transmembrane domain F / TM (SEQ ID No. 4 to 18) or heterologous transmembrane domain, such as the transmembrane domain gp160 / TM of the HIV-1 virus gp160 protein (SEQ ID NO. 179), the transmembrane domain HA / TM of the influenza virus HA protein (SEQ ID NO. 19), the transmembrane domain S / TM of the SARS-CoV-2 S protein (SEQ ID NO. 181), and other transmembrane domains.

[0015] Furthermore, in a feasible implementation, the C-terminus of the homologous transmembrane domain of the RSV F protein also includes an intracellular domain of the RSV F protein, which may be the full-length intracellular domain of the RSV F protein (such as the amino acid sequence of SEQ ID No. 228 or SEQ ID No. 232) or a truncated intracellular domain (such as the amino acid sequence lysine-alanine (KA), SEQ ID No. 225 to 227, or SEQ ID No. 229 to 231).

[0016] Furthermore, in a feasible implementation, the C-terminus of the heterologous transmembrane domain also includes the intracellular domain of the heterologous protein.

[0017] Further feasiblely, the F protein-based RSV antigen also comprises a signal peptide; preferably, the signal peptide is located at the N-terminus; preferably, the signal peptide has an amino acid sequence as shown in any one of SEQ ID No. 26 to 27 and 172 to 178.

[0018] Further feasiblely, the above-mentioned structural domains can be directly connected or indirectly connected through connectors or spacer sequences; available connectors include, but are not limited to, SEQ ID No. 182 or 183.

[0019] In a preferred embodiment, the F protein-based RSV antigen has an amino acid sequence selected from the following:

[0020] SEQ ID No. 32 to 40, 47 to 55, 58 to 65, 184, 185, 205, 208, 214, 220.

[0021] Secondly, this application provides a polynucleotide that encodes the F protein-based RSV antigen as described in the first aspect above.

[0022] In a specific implementation, the polynucleotide can be DNA or mRNA; preferably, the polynucleotide is a nucleotide sequence optimized with human codons.

[0023] In some embodiments, the polynucleotide is a DNA molecule; preferably, the DNA molecule comprises, or is composed of, a DNA sequence as shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221.

[0024] In other embodiments, the polynucleotide is an mRNA molecule; preferably, the mRNA molecule comprises, or is composed of, an RNA sequence transcribed from a DNA sequence as shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221.

[0025] 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.

[0026] In some embodiments, the nucleic acid construct comprises, or is composed of, a DNA sequence as shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, and 221.

[0027] In other embodiments, the nucleic acid construct comprises, or is composed of, a DNA sequence as shown in any one of SEQ ID Nos. 118 to 126, 133 to 141, 144 to 151, 190, 191, 207, 210, 216, 222.

[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 multimer of an RSV antigen based on the F protein, the multimer comprising the RSV antigen based on the F protein as described in the first aspect above, preferably a trimer, hexamer, or nonamer.

[0034] In a seventh aspect, this application provides the use of the RSV antigen based on the F protein 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 antigen multimer as described in the sixth aspect above in the preparation of vaccines / medications for the prevention and / or treatment of respiratory syncytial virus infection.

[0035] In a feasible implementation, the vaccine may contain the RSV antigen based on the F protein 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 antigen multimer as described in the sixth aspect above as the sole antigenic component.

[0036] In other feasible implementations, in addition to the F protein-based RSV antigen 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 antigen multimer as described in the sixth aspect above, the vaccine may also contain immunogenic components derived from other pathogens.

[0037] Eighthly, this application provides an immunogenic composition or vaccine comprising an RSV antigen based on the F protein 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 antigen multimer as described in the sixth aspect above, and physiologically or pharmaceutically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

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

[0039] Optionally, 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.

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

[0041] (1) Eukaryotic expression vectors; and

[0042] (2) Construct a DNA sequence encoding the RSV antigen based on the F protein as described in the first aspect above, which is inserted into the eukaryotic expression vector;

[0043] Optionally, the DNA sequence is any one of the DNA sequences shown in SEQ ID No. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, 118 to 126, 133 to 141, 144 to 151, 190, 191, 207, 210, 216, 222;

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

[0045] In some other preferred embodiments, the immunogenic composition or vaccine is a respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising:

[0046] (I) The mRNA sequence encoding the F protein-based RSV antigen as described in the first aspect above; and

[0047] (II) Delivery carrier.

[0048] Optionally, the mRNA sequence is an mRNA sequence transcribed from a DNA sequence as shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, 118 to 126, 133 to 141, 144 to 151, 190 to 192, 206, 207, 209, 210, 215, 216, 221, and 222;

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

[0051] In some other preferred embodiments, the immunogenic composition or vaccine is a respiratory syncytial virus-virus vector vaccine, comprising:

[0052] (1) Viral backbone vector; and

[0053] (2) Constructing a DNA sequence encoding the RSV antigen based on the F protein as described in the first aspect above into the viral backbone vector;

[0054] Optionally, the DNA sequence is any one of the DNA sequences shown in SEQ ID No. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, 118 to 126, 133 to 141, 144 to 151, 190, 191, 206, 207, 209, 210, 215, 216, 221, 222;

[0055] 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.

[0056] In some other preferred embodiments, the immunogenic composition or vaccine is a respiratory syncytial virus nanoparticle vaccine, comprising an RSV vaccine based on the F protein and a nanoparticle carrier as described in the first aspect above.

[0057] Optionally, the nanoparticle carrier is ferritin. The RSV vaccine based on F protein as described in the first aspect above is covalently linked to ferritin and self-assembled into nanoparticles, so that the RSV vaccine is presented on the surface of the nanoparticles.

[0058] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, an oral formulation, or a parenteral formulation;

[0059] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0060] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0061] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

[0062] Ninthly, this application provides a method of administration for the above-mentioned immunogenic composition or vaccine, which can be administered orally, nasally, intramuscularly, subcutaneously, intradermally, or intravenously.

[0063] 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: an RSV antigen based on the F protein 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, a polymer of the RSV antigen based on the F protein as described in the sixth aspect above, and / or a vaccine or immunogenic composition as described in the eighth aspect above.

[0064] 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.

[0065] In one aspect, this application provides a multivalent immunogenic composition or vaccine comprising, as described in the first aspect above, an RSV antigen based on the F protein, 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, or an RSV antigen multimer based on the F protein as described in the sixth aspect, and immunogenic components derived from other pathogens.

[0066] In feasible implementation schemes, the immunogenic component derived from other pathogens is selected from at least one antigen selected from influenza virus antigen, parainfluenza virus antigen, human metapneumovirus antigen, rhinovirus antigen, adenovirus antigen, bocavirus antigen, coronavirus antigen, novel coronavirus antigen, pneumococcal antigen, mycoplasma antigen, diphtheria toxoid, tetanus toxoid, pertussis vaccine, Haemophilus influenzae vaccine, interstitial lung virus antigen, Neisseria meningitidis vaccine, polio vaccine, or hepatitis vaccine. Beneficial effects

[0067] This application designs an RSV antigen based on the F protein by introducing a trimerizing domain at a specific site in the F protein before RSV fusion, while retaining the homologous transmembrane domain of the RSV F protein or introducing a heterologous transmembrane domain. This design improves the interaction between the trimerizing domain and the transmembrane domain, making its pre-fusion conformation more stable and enhancing the stability of the resulting trimerized protein. This, in turn, enhances the immunogenicity of the RSV F protein and improves its immune response. The RSV antigen based on the F protein of this application 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).

[0068] 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 F protein-based RSV antigen of this application has broad applicability. Detailed Implementation

[0069] To better understand this application, definitions and explanations of relevant terms are provided below.

[0070] 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.

[0071] RSVF 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, RSVF 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 protease 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 RSVF extracellular domain, which includes the F2 protein and the F1 extracellular domain.

[0072] RSVF 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 RSVF 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 RSV wild-type F protein sequence or F0 polypeptide sequence shown in SEQ ID NO:28-31.

[0073] 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.

[0074] "PreF1" and "PreF4" refer to mutants that have undergone pre-fusion conformational stabilization modification on the wild-type F protein of RSVA2 and RSVTX-79233 strains, respectively.

[0075] 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 a polypeptide that facilitates the assembly of the polypeptide into a trimer. For instance, a trimerizing domain can promote trimer assembly 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. The term is also used to refer to polynucleotides encoding such peptides or polypeptides. Each domain may contain a linker / spacer sequence or part of another domain without affecting its primary physiological function. For example, the extracellular domain of an F protein may contain part of a transmembrane domain without affecting the extracellular domain's role as a primary target region for antibody binding.

[0076] The terms "linker," "connector," or "spacer" are natural or artificial bifunctional molecules that can be used to link two or more 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, or other sequences that can serve a linking or spacer function. 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) that results in the formation of a continuous polypeptide chain from the first and second polypeptides. 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.

[0077] The term "mutant" refers to a molecule whose amino acid sequence differs from the native 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 native or reference sequence. Typically, variants share at least 50% homology with the native or reference sequence. In some embodiments, variants share at least 80% or at least 90% homology with the native or reference sequence.

[0078] 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.

[0079] 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.

[0080] 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).

[0081] 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.

[0082] "Pathogen" is a collective term for microorganisms and parasites that can cause disease. Microorganisms constitute the vast majority, including viruses, chlamydia, rickettsiae, mycoplasma, bacteria, spirochetes, and fungi; parasites mainly include protozoa and worms. Pathogens are parasitic organisms, and their natural hosts are animals, plants, and humans. There are more than 400 species of microorganisms that can infect humans, and they are widely present in the human mouth, nose, pharynx, digestive tract, urogenital tract, and skin.

[0083] "Antigen" refers to a compound, composition, or substance that can stimulate antibody production and / or T cell response in an animal or human body, including compositions introduced into an animal or human body 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).

[0084] 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.

[0085] "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.

[0086] "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.

[0087] "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.

[0088] 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.

[0089] "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).

[0090] "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.

[0091] 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.

[0092] "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.

[0093] 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.

[0094] 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.

[0095] 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. Attached Figure Description

[0096] 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.

[0097] Figure 1 shows the composition of the respiratory syncytial virus antigen of this application.

[0098] Figure 2 shows the composition of the control antigen of this application.

[0099] Figure 3 shows a schematic diagram of the mRNA composition described in the embodiments.

[0100] Figure 4 shows the immunogenicity of the mRNA vaccine with phage T4 minor fibrin folds inserted at different positions in the RSVA-type F protein in Example 4 in mice. Figure A shows a schematic diagram of the RSV F protein antigen with phage T4 minor fibrin folds inserted at different positions in the PreF1 protein. Figure B shows the neutralizing antibody titers induced by the RSVA-type F protein antigen mRNA vaccine. The bar chart shows the geometric mean titer (GMT) of neutralizing antibody NT50 in each group of 6 mice (n=6). The GMT values ​​are labeled at the top of the bars, and the error bars represent the 95% CI (confidence interval).

[0101] Figure 5 shows the immunogenicity of the mRNA vaccines with and without transmembrane domain-connected RSVA type F protein antigens in mice, as described in Example 4. Figure A shows a schematic diagram of RSVA type F protein antigens with and without transmembrane domain-connected PreF1 protein. Figure B shows the neutralizing antibody titers induced by the RSVA type F protein antigen mRNA vaccine. The neutralizing antibody titers are presented as a bar chart of the geometric mean titer (GMT) of NT50 for each group of 6 mice, with the GMT values ​​labeled at the top of the bars. The error bars represent the 95% CI (confidence interval).

[0102] Figure 6 shows the immunogenicity of the mRNA vaccine containing RSVA-type F protein antigens with different transmembrane domain lengths in Example 4 in mice. Figure A shows a schematic diagram of RSVA-type F protein antigens with different transmembrane domain lengths connected in the PreF1 protein. Figure B shows the neutralizing antibody titers induced by the RSVA-type F protein antigen mRNA vaccine. The bar chart shows the geometric mean titer (GMT) of neutralizing antibody NT50 in each group of 6 mice (n=6). The GMT values ​​are labeled at the top of the bars, and the error bars represent the 95% CI (confidence interval).

[0103] Figure 7 shows the immunogenicity of the mRNA vaccine containing RSVA-type F protein antigens with different trimerization and transmembrane domains inserted in Example 4 in mice. Figure A shows a schematic diagram of RSV F protein antigens with different trimerization and transmembrane domains inserted into the PreF1 protein. Figure B shows the neutralizing antibody titers induced by the RSVA-type F protein antigen mRNA vaccine. The bar chart shows the geometric mean titer (GMT) of neutralizing antibody NT50 in each group of 6 mice (n=6). The GMT values ​​are labeled at the top of the bars, and the error bars represent the 95% CI (confidence interval).

[0104] Figure 8 shows the immunogenicity of the RSVA-type F protein antigen mRNA vaccine, which connects the transmembrane and intracellular domains, in mice in Example 4. Figure A shows a schematic diagram of the RSVA-type F protein antigen connecting the transmembrane and intracellular domains in the PreF1 protein. Figure B shows the neutralizing antibody titers induced by the RSVA-type F protein antigen mRNA vaccine. The bar chart shows the geometric mean titer (GMT) of neutralizing antibody NT50 in each group of 6 mice (n=6). The GMT values ​​are labeled at the top of the bars, and the error bars represent the 95% CI (confidence interval).

[0105] Figure 9 shows the immunogenicity of the mRNA vaccine in mice with RSVB type F protein antigens containing trimerizing domains at different positions, with different trimerizing domains inserted, and with and without transmembrane domains connected, as described in Example 4. Figure A shows a schematic diagram of RSVB type F protein antigens in the PreF4 protein with RSVB type F protein antigens containing trimerizing domains at different positions, with different trimerizing domains inserted, and with and without transmembrane domains connected. Figure B shows the neutralizing antibody titers induced by the mRNA vaccine containing RSVB type F protein antigens. The bar chart shows the geometric mean titer (GMT) of neutralizing antibody NT50 in each group of 6 mice (n=6). The GMT values ​​are labeled at the top of the bars, and the error bars represent the 95% CI (confidence interval).

[0106] Figure 10 shows the immunogenicity of the RSVA-type F protein antigen mRNA vaccine in mice, with the transmembrane domain located at the C-terminus or N-terminus of the trimerization domain, in Example 4. Figure A shows a schematic diagram of RSV F protein antigen with the transmembrane domain added to the C-terminus or N-terminus of the phage T4 minor fibrin fold. Figure B shows the neutralizing antibody titers induced by the RSVA-type F protein antigen mRNA vaccine. The bar chart shows the geometric mean titer (GMT) of neutralizing antibody NT50 in each group of 6 mice (n=6). The GMT values ​​are labeled at the top of the bars, and the error bars represent the 95% CI (confidence interval).

[0107] Detailed Implementation

[0108] Example 1: Structural design of respiratory syncytial virus antigen based on F protein

[0109] In this embodiment, the following structures of the anti-respiratory syncytial virus antigen of this application were designed.

[0110] Figure 1 shows the composition of the antigen of this application; Tables 1 and 2 show the amino acid sequence and composition of the antigen of this application. The anti-respiratory syncytial virus antigen is composed of an extracellular domain, a trimerizing domain, a transmembrane domain, and an intracellular domain linked together in a direction from the N-terminus to the C-terminus. Different domains can be directly linked or indirectly linked through linkers or spacer sequences.

[0111] Figure 2 shows the composition of the control antigen of this application; Tables 3 and 4 show the amino acid sequence and composition of the control antigen of this application. The control antigen is composed of an extracellular domain linked to a trimerizing domain, or an extracellular domain linked to a transmembrane domain, or an extracellular domain, a transmembrane domain, and a trimerizing domain linked in series from the N-terminus to the C-terminus. Different structural domains can be directly linked or indirectly linked through linkers or spacer sequences.

[0112] The sequences of the above antigens do not contain their endogenous signal peptides. The endogenous signal peptide sequences of the PreF1-related antigen and the control antigen are shown in SEQ ID NO: 26, and the endogenous signal peptide sequences of the PreF4-related antigen and the control antigen are shown in SEQ ID NO: 27.

[0113] Table 1. Antigen composition reconstructed from extracellular domain-trimerizing domain-transmembrane domain

[0114] Table 2. Antigen composition reconstructed from extracellular domain-trimerizing domain-transmembrane domain-intracellular domain

[0115] Table 3. Composition of Control Antigen 1

[0116] Table 4. Composition of Control Antigen 2

[0117] Example 2: Preparation of respiratory syncytial virus antigen mRNA and packaging of lipid nanoparticles (LNP) according to this application

[0118] A pUC57 plasmid containing the T7 promoter (SEQ ID NO: 161), 5'UTR (SEQ ID NO: 162), coding sequences (CDS) of each reconstructed polypeptide, 3'UTR (SEQ ID NO: 163), polyadenylate tail (SEQ ID NO: 164), 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). Capping rate and polyadenylate tail of mRNA were analyzed by liquid chromatography-mass spectrometry (LC-MS). The pH of the mRNA solution, residual DNA, protein, and double-stranded RNA impurities were also analyzed. Figure 3 shows a schematic diagram of the mRNA composition.

[0119] 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.

[0120] Using the above procedure, mRNA-LNPs corresponding to each antigen shown in Example 1 were prepared as mRNA vaccines.

[0121] Example 3: Immunization and Sample Collection of Laboratory Animals

[0122] 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.

[0123] Example 4: Plaque Reduction Neutralization Assay (FRNT) for detecting neutralizing antibody titers in the serum of immunized mice

[0124] Serum neutralizing antibody titers (NT50) were detected using the focal reduction neutralization assay (FRNT). Neutralizing antibodies in the PreF1-associated construct were detected using RSV-A2-GFP virus, and neutralizing antibodies in the PreF4-associated construct were detected using RSV-B1-GFP virus.

[0125] HEP-2 cells (purchased from ATCC, DMEM medium + 10% FBS) 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 the type A strain (PreF1-related reconstructed peptide) and neutralizing antibodies were detected using RSV-A2-GFP virus; the sequence was derived from the type B strain (PreF4-related reconstructed peptide) 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 the serum-virus mixture to a 96-well plate of HEP-2 cells: Remove the cell culture supernatant, add 200 μL of the neutralized serum-virus mixture to each well, and perform two replicates for each serum dilution. Incubate the cell culture plate at 37°C for 48 h. Fluorescent spot reading: Remove the culture medium, wash the cell surface twice with PBS, pat dry, and read the spot count using an ELISA reader. Finally, calculate the RSV neutralizing antibody titer by combining the virus titer and serum dilution factor.

[0126] The results and analysis are as follows.

[0127] Figure 4A shows a schematic diagram of RSV antigens inserted at different positions in the PreF1 protein by the phage T4 minor fibrin fold. Figure 4B shows the neutralizing antibody titers induced by mRNA vaccines corresponding to antigens inserted at different positions in the PreF1 protein by the phage T4 minor fibrin fold, indicating that the phage T4 minor fibrin fold inserted into the PreF1 protein at 514 (PreF1) 514 -T4-F / TM A515-550 ), 516 (PreF1) 516 -T4-F / TM A517-550 ), 519 (PreF1) 519 -T4-F / TM A520-550 ), 522 (PreF1) 522 -T4-F / TM A523-550 ) and 524 (PreF1) 524 -T4-F / TM A525-550 The amino acid sequence following the 5th amino acid can induce very high neutralizing antibody titers, while the phage T4 minor fibrin fold inserts into the 528th fold of the PreF1 protein (PreF1...). 528 -T4-F / TM A529-550 ), 532 (PreF1) 532 -T4-F / TM A533-550 ), 536 (PreF1) 536 -T4-F / TM A537-550 The titer of neutralizing antibodies induced after amino acids 514, 516, 519, 522, 528, 532, and 536 of the PreF1 protein was lower. The neutralizing antibody titers induced by mRNA vaccines corresponding to antigens from phage T4 minor fibrin folds inserted after amino acids 514, 516, 519, 522, 528, 532, and 536 of the PreF1 protein were respectively those of PreF1. 524 -T4-F / TM A525-550 The values ​​were 1.85, 1.38, 1.95, 1.07, 0.52, 0.29, and 0.32 times higher. These results indicate that amino acid sites 514 to 524 of the PreF protein are suitable for the insertion of the trimerization domain.

[0128] Figure 5 shows the neutralizing antibody titers induced by mRNA vaccines corresponding to RSVA-type F protein antigens with and without the C-terminus of the trimer domain connected to the transmembrane domain. It shows the antigen protein PreF1 with the C-terminus of the trimer domain connected to the transmembrane domain. 514 -T4-F / TM A515-550 PreF1 519 -T4-F / TM A520-550 PreF1 524 -T4-F / TM A525-550It can induce very high neutralizing antibody titers, where the neutralizing antibody titers are respectively the antigen protein PreF1 whose C-terminus of the trimer domain is not connected to the transmembrane domain. 514 -T4、PreF1 519 -T4、PreF1 524 -T4-induced neutralizing antibody titers were 1.86-fold, 2.04-fold, and 1.50-fold higher. The antigen protein PreF1, whose C-terminus of the trimerization domain is linked to a heterologous transmembrane domain (the transmembrane domain of the influenza virus HA protein), exhibits these effects. 519 -T4-HA / TM-induced neutralizing antibodies are PreF1 antigen proteins that do not have a transmembrane domain. 519 -T4 was 1.39 times that of T4. The above results indicate that antigen proteins with a C-terminus of the trimerization domain connected to a transmembrane domain (either a homologous or heterologous transmembrane domain of the RSV F protein) are superior to antigen proteins without a connected transmembrane domain.

[0129] Figure 6 shows the neutralizing antibody titers induced by mRNA vaccines corresponding to RSVA-type F protein antigens with C-terminal trimerization domains linked to transmembrane domains of different lengths. It illustrates the antigen proteins PreF1 with C-terminal trimerization domains linked to transmembrane domains of different lengths. 519 -T4-F / TM A515-550 PreF1 519 -T4-F / TM A520-550 PreF1 519 -T4-F / TM A525-550 The induced neutralizing antibody titers were for the antigen protein PreF1, which does not have a transmembrane domain. 519 -T4 titers were 1.91, 2.04, and 1.93 times higher. These results indicate that linking transmembrane domains of different lengths to the C-terminus of the trimerization domain can increase the neutralizing antibody titer.

[0130] Figure 7 shows the neutralizing antibody titers induced by mRNA vaccines corresponding to RSVA-type F protein antigens with or without transmembrane domains attached to the C-terminus of antigen proteins with different trimerization domains. It illustrates the PreF1 antigen protein with a transmembrane domain attached to the C-terminus of the phage T4 minor fibrin fold. 514 -T4-F / TM A515-550 The induced neutralizing antibody is the antigen protein PreF1, which is not linked to the transmembrane domain. 514 -1.81 times that of T4. The antigen protein PreF1, which connects to the transmembrane domain, is linked at the C-terminus of the isoleucine zipper IZ short chain trimerization domain. 514 -IZ-F / TM A515-550 PreF1 519 -IZ-F / TM A525-550 The induced neutralizing antibodies were PreF1 antigen proteins that did not have a transmembrane domain attached. 514-IZ was 6.22 times and 6.82 times higher. The antigen protein PreF1, which crosses the membrane domain, is linked to the C-terminus of the MTQ trimerization domain. 514 -MTQ-F / TM A515-550 PreF1 519 -MTQ-F / TM A525-550 The induced neutralizing antibodies were PreF1 antigen proteins that did not have a transmembrane domain attached. 514 -MTQ titers were 2.49 times and 4.22 times higher. These results indicate that further linking a transmembrane domain to the C-terminus of different trimerization domains can improve neutralizing antibody titers.

[0131] Figure 8 shows the neutralizing antibody titers induced by mRNA vaccines corresponding to the RSVA-type F protein antigen that connects the transmembrane and intracellular domains. It is shown as the antigen protein PreF1, which connects the transmembrane and intracellular domains at the C-terminus of the phage T4 minor fibrin trimer. 519 -T4-F / TM A525-552 PreF1 519 -T4-F / TM A525-565 PreF1 519 -T4-F / TM A525-574 The induced neutralizing antibody titers were for the antigen protein PreF1, which lacks a transmembrane domain and an intracellular domain. 519 -T4 titers were 1.88, 1.51, and 1.33 times higher. These results indicate that further connecting the transmembrane and intracellular domains to the trimerization domain can improve the neutralizing antibody titer.

[0132] Figure 9 shows the neutralizing antibody titers induced by mRNA vaccines corresponding to RSV type B F protein antigens with different insertion positions of the trimerizing domain, different insertion positions of the trimerizing domain, and with and without the transmembrane domain. It demonstrates the presence of the antigen protein PreF4 with the transmembrane domain linked to the C-terminus of the trimerizing domain, based on the insertion of the phage T4 minor fibrin fold trimerizing domain at different positions. 514 -T4-F / TM B525-550 PreF4 519 -T4-F / TM B525-550 The induced neutralizing antibody titers were for the antigen protein PreF4, which does not have a transmembrane domain. 514 -T4、PreF4 519 -1.52 and 1.39 times that of T4. The antigen protein PreF4, which links to the transmembrane domain, is located at the C-terminus of the MTQ trimerization domain. 519 -MTQ-F / TM B520-550 The induced neutralizing antibody is the antigen protein PreF4, which lacks a transmembrane domain. 519-1.41 times that of MTQ. The above results indicate that antigen design that further links the transmembrane domain to the C-terminus of the trimerization domain can improve the neutralizing antibody titer of RSVB type antigens.

[0133] Figure 10 shows the neutralizing antibody titers induced by mRNA vaccines corresponding to RSVA-type F protein antigens with transmembrane domains linked to the C-terminus or N-terminus of the phage T4 minor fibrin trimer domain. It shows the antigen protein PreF1 with its transmembrane domain linked to the C-terminus of the phage T4 minor fibrin trimer domain. 514 -T4-F / TM A515-550 PreF1 519 -T4-F / TM A520-550 PreF1 524 -T4-F / TM A525-550 The induced neutralizing antibody titers were determined by the addition of the transmembrane domain of the antigen protein PreF1 to the N-terminus of the trimerization domain of the phage T4 minor fibrin fold. 550 -T4 titers were 1.91, 2.00, and 1.78 times higher. These results indicate that linking a transmembrane domain to the C-terminus of the trimerization domain can increase the neutralizing antibody titer. Industrial applicability

[0134] This application designs an RSV antigen based on the F protein by introducing a trimerizing domain at a specific site in the F protein before RSV fusion, while retaining the homologous transmembrane domain of the RSV F protein or introducing a heterologous transmembrane domain. This design improves the interaction between the trimerizing domain and the transmembrane domain, making its pre-fusion conformation more stable and enhancing the stability of the resulting trimerized protein. This, in turn, enhances the immunogenicity of the RSV F protein and improves its immune response. The RSV antigen based on the F protein of this application 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.

[0135] 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 F protein-based RSV antigen of this application has broad applicability.

Claims

1. [Correction 24.02.2026 based on Rule 91] A respiratory syncytial virus (RSV) antigen based on the F protein, characterized in that, The antigen comprises the following domains sequentially linked from the N-terminus to the C-terminus: the extracellular domain, trimerization domain, and transmembrane domain of the RSV F protein; wherein the trimerization domain is inserted at any of amino acid positions 514 to 524 of the wild-type RSV F protein sequence as shown in SEQ ID No. 28 to 31, or at a corresponding position thereof. The transmembrane domain is either a homologous or heterologous transmembrane domain of the RSV F protein.

2. [Correction 24.02.2026 according to Rule 91] The RSV antigen based on the F protein according to claim 1 is characterized in that, The F protein is selected from the F protein of type A or type B RSV virus, or a mutant that has at least 80% amino acid 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, isoleucine zipper long chain, isoleucine zipper short chain, leucine zipper GCN4 and its variants, pulmonary surfactant-associated protein D trimer domain, collagen trimer domain, cartilage matrix protein-1 long chain trimer domain or cartilage matrix protein-1 short chain trimer domain; And / or, the heterologous transmembrane domain is selected from: the transmembrane domain of the HIV-1 virus gp160 protein, the transmembrane domain of the influenza virus HA protein, or the transmembrane domain of the novel coronavirus S protein.

3. [Correction 24.02.2026 according to Rule 91] The RSV antigen based on the F protein according to claim 1 is characterized in that, The trimerization domain has an amino acid sequence selected from any of the following: sequences shown in SEQ ID No. 1 to SEQ ID No. 3 or SEQ ID No. 165 to 171; And / or, the homologous transmembrane domain of the F protein has an amino acid sequence as shown in any one of SEQ ID No. 4 to SEQ ID No. 15; And / or, the heterologous transmembrane domain has an amino acid sequence selected from any one of SEQ ID No. 19, 179, 181.

4. [According to Rule 91, amended 24.02.2026] The RSV antigen based on the F protein according to any one of claims 1 to 3, characterized in that, The C-terminus of the antigen further includes the intracellular domain of the F protein.

5. [According to Rule 91, Amendment 24.02.2026] A polynucleotide encoding the F protein-based RSV antigen as described in any one of claims 1 to 4.

6. [Correction 24.02.2026 according to Rule 91] The polynucleotide according to claim 5, characterized in that, The polynucleotide is a DNA molecule or an RNA molecule, preferably an mRNA molecule.

7. [Correction 24.02.2026 according to Rule 91] The polynucleotide according to claim 6, characterized in that, The polynucleotide has a DNA sequence or a transcribed RNA sequence thereof shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, or is composed of such sequences.

8. [Correction 24.02.2026 based on Rule 91] A nucleic acid construct, characterized in that, It comprises the polynucleotide as described in any one of claims 5 to 7, and at least one expression regulatory element operatively linked to the polynucleotide.

9. [Correction 24.02.2026 according to Rule 91] The nucleic acid construct according to claim 8, characterized in that, The nucleic acid construct comprises, or consists of, any of the sequences shown in SEQ ID Nos. 118 to 126, 133 to 141, 144 to 151, 190, 191, 207, 210, 216, 222, or their transcribed RNA sequences.

10. [Revised according to Rule 91, 24.02.2026] An expression vector comprising the nucleic acid construct as claimed in any one of claims 8 or 9.

11. [Revised according to Rule 91, 24.02.2026] A host cell wherein a polynucleotide as described in any one of claims 5 to 7, a nucleic acid construct as described in any one of claims 8 or 9, or an expression vector as described in claim 10 has been transformed or transferred.

12. [According to Rule 91, Amendment 24.02.2026] The multimer of the RSV antigen based on the F protein according to any one of claims 1 to 4.

13. [Correction 24.02.2026 according to Rule 91] The polymer according to claim 12, characterized in that... The polymer is a trimer, hexamer, or nonamer.

14. [Correction 24.02.2026 according to Rule 91] The polymer according to any one of claim 12 or 13, characterized in that... The polymer is a homopolymer or a heteropolymer.

15. [Correction 24.02.2026 according to Rule 91] The use of the F protein-based RSV antigen as claimed in any one of claims 1 to 4, the polynucleotide as claimed in any one of claims 5 to 7, the nucleic acid construct as claimed in any one of claims 8 to 9, the expression vector as claimed in claim 10, or the polymer as claimed in any one of claims 12 to 14 in the preparation of vaccines / medications for the prevention and / or treatment of respiratory syncytial virus infection.

16. [Correction 24.02.2026 according to Rule 91] The application according to claim 15 is characterized in that, The vaccine also contains other immunogenic components.

17. [Revised according to Rule 91, 24.02.2026] An immunogenic composition or vaccine comprising an RSV antigen based on the F protein as claimed in any one of claims 1 to 4, a polynucleotide as claimed in any one of claims 5 to 7, a nucleic acid construct as claimed in any one of claims 8 to 9, an expression vector as claimed in claim 10, or a polymer as claimed in any one of claims 12 to 14, and a physiologically or pharmaceutically acceptable adjuvant, excipient, diluent, carrier, or medium.

18. [Revised according to Rule 91, 24.02.2026] The immunogenic composition or vaccine according to claim 17 is a respiratory syncytial virus protein or polypeptide vaccine comprising the RSV antigen based on the F protein according to any one of claims 1 to 4 or the polymer and adjuvant according to any one of claims 12 to 14.

19. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 18 is 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.

20. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 17 is a respiratory syncytial virus DNA vaccine, said DNA vaccine comprising: (a) A eukaryotic expression vector comprising a DNA sequence encoding an RSV antigen based on the F protein as described in any one of claims 1-4; and (b) DNA vaccine adjuvants.

21. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 20, which is a respiratory syncytial virus DNA vaccine, is characterized in that... The DNA sequence encoding the F protein-based RSV antigen as described in any one of claims 1 to 4 is the DNA sequence shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, 118 to 126, 133 to 141, 144 to 151, 190, 191, 207, 210, 216, 222; 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 one or more of the following: human granulocyte-macrophage community-stimulating factor, human interleukin-12, or CpG oligonucleotides.

22. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to any one of claim 20 or 21, which is a respiratory syncytial virus DNA vaccine, characterized in that... The DNA vaccine further includes a delivery vector, which is selected from one or more of lipid nanoparticles, cationic nanoemulsions, peptides, polymers, cationic peptide polymers, and cationic peptide lipid nanoparticles.

23. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 17 is a respiratory syncytial virus mRNA vaccine, said mRNA vaccine comprising: (a) An mRNA sequence encoding the RSV antigen based on the F protein as described in any one of claims 1-4; (b) Delivery carrier; and / or (c) mRNA vaccine adjuvants.

24. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 23, which is a respiratory syncytial virus mRNA vaccine, is characterized in that... The mRNA sequence encoding the F protein-based RSV antigen as described in any one of claims 1 to 4 is an mRNA sequence transcribed from a DNA sequence as shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, 118 to 126, 133 to 141, 144 to 151, 190, 191, 207, 210, 216, 222; And / or, the mRNA vaccine adjuvant is selected from: lipid nanoparticles, emulsions, Toll-like receptor agonists and / or CpG oligonucleotides; And / or, the delivery carrier is selected from one or more of lipid nanoparticles, cationic nanoemulsions, peptides, polymers, cationic peptide polymers, and cationic peptide lipid nanoparticles.

25. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 17, which is a respiratory syncytial virus-virus vector vaccine, comprises: (a) Viral backbone vector; and (b) A DNA sequence encoding the F protein-based RSV antigen as described in any one of claims 1 to 4, constructed into the viral backbone vector.

26. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 25, which is a respiratory syncytial virus-virus vector vaccine, is characterized in that, The DNA sequence encoding the F protein-based RSV antigen as described in any one of claims 1 to 4 is a DNA sequence as shown in any one of SEQ ID Nos. 75 to 83, 90 to 98, 101 to 108, 187, 188, 206, 209, 215, 221, 118 to 126, 133 to 141, 144 to 151, 190, 191, 207, 210, 216, 222; 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. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to any one of claims 17 to 26 is characterized in that, It can be administered orally, nasally, intramuscularly, subcutaneously, intradermally, or intravenously.

28. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to any one of claims 17 to 26 is characterized in that, The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, or parenteral formulation.

29. [Correction 24.02.2026 according to Rule 91] The immunogenic composition or vaccine according to claim 28 is characterized in that, The nasal spray is selected from aerosols, sprays, or 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.

30. [Correction 24.02.2026 based on Rule 91] A multivalent immunogenic composition or vaccine, characterized in that, The multivalent immunogenic composition or vaccine comprises the F protein-based RSV antigen as described in any one of claims 1 to 4, the polynucleotide as described in any one of claims 5 to 7, the nucleic acid construct as described in any one of claims 8 to 9, the expression vector as described in claim 10, or the polymer as described in any one of claims 12 to 14, and immunogenic components derived from other pathogens.

31. [Correction 24.02.2026 according to Rule 91] The multivalent immunogenic composition or vaccine according to claim 30 is characterized in that, Immunogenic components derived from other pathogens are selected from at least one antigen from the following: influenza virus antigen, parainfluenza virus antigen, human metapneumovirus antigen, rhinovirus antigen, adenovirus antigen, bocavirus antigen, coronavirus antigen, novel coronavirus antigen, pneumococcal antigen, mycoplasma antigen, diphtheria toxoid, tetanus toxoid, pertussis vaccine, Haemophilus influenzae vaccine, interstitial lung virus antigen, Neisseria meningitidis vaccine, polio vaccine, or hepatitis vaccine.

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