Mutant RSV protein

Abstract

The present invention relates to a recombinant Respiratory Syncytial Virus (RSV) F protein, an isolated nucleic acid molecule encoding the recombinant RSV F protein, a vector carrying the nucleic acid molecule, and a host cell. The invention also relates to a method for producing the recombinant RSV F protein. Furthermore, the invention relates to an immunogenic composition comprising the recombinant RSV F protein or the nucleic acid molecule encoding the recombinant RSV F protein.

Description

mutant RSV proteins

[0001] The present invention relates to a recombinant respiratory syncytial virus (RSV) F protein, an isolated nucleic acid molecule encoding the recombinant RSV F protein, a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule. The present invention also relates to a method for producing the recombinant RSV F protein. The present invention also relates to an immunogenic composition comprising the recombinant RSV F protein or a nucleic acid molecule encoding the recombinant RSV F protein.

[0002] Respiratory syncytial virus (RSV) is a negative-sense, single-stranded, non-segmented RNA virus belonging to the family Paramyxoviridae. RSV causes infections of the lower and upper respiratory tract in humans, primarily affecting infants and young children, but can also cause life-threatening respiratory disease in immunocompromised individuals and the elderly.

[0003] Currently, nirsevimab and palivizumab are used as preventive monoclonal antibody injections for infants and young children to reduce the risk of severe RSV, and Arexvy developed by GSK is an RSV subunit vaccine for elderly people, Abrysvo developed by Pfizer is an RSV subunit vaccine for elderly people and pregnant women, and mRESVIA developed by Moderna is an RSV mRNA vaccine for elderly people, each of which has been approved by the U.S. Food and Drug Administration (FDA). These two antibody injections target the F protein of RSV, and all three vaccines contain the stabilized prefusion recombinant F protein of RSV or its mRNA as an antigen.

[0004] The F protein of RSV is a fusion glycoprotein abundant in the envelope, existing on the viral surface in a trimeric "pre-fusion" state. Upon entry, it inserts its hydrophobic fusion loop into the cell membrane and undergoes extensive conformational changes to fold into an energetically favorable trimeric "post-fusion" state, fusion of the viral and cell membranes.

[0005] The F protein of RSV is initially expressed as a single 574-residue polypeptide precursor (“F0” or “F0 precursor”) containing an N-terminal signal peptide sequence (residues 1-25) and an ectodomain (ED) containing three heptad repeats (HRA, HRB and HRC), a transmembrane domain (TM) and a cytoplasmic tail (CT). During translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum, and the remainder of the F0 precursor (i.e., residues 26-574) is further cleaved by a cellular protease (furin) at two polybasic sites (residues 109 / 110 and 136 / 137), removing the 27-amino acid intervening sequence designated p27 (residues 110-136) and forming the RSV F protomer, a heterodimer of F1 (residues 137-574) and F2 (residues 26-109) linked together via two disulfide bonds. The three protomers assemble to ultimately form the RSV F protein complex (a homotrimer of the three protomers).

[0006] RSV F protein stabilized in the pre-fusion conformation has been shown to elicit greater neutralizing immune responses in animal models than those observed with the post-fusion conformation of RSV F protein. For example, “DS-Cav1,” a recombinant RSV F protein stabilized in the pre-fusion conformation containing the S155C, S290C, S190F, and V207L ​​substitutions, was reported to elicit neutralizing immune responses several times greater than those observed with the post-fusion conformation of RSV F protein (McLellan et al., Science (2013) vol. 342, p. 592-598; International Patent Publication No. WO2014160463). In addition, the second-generation antigen "DS2" variants, which improved the structural stability and immunoprotective response of "DS-Cav1", were reported to have genetically linked F subunits, fusion peptide deletions, and interprotomeric shifts stabilized by additional disulfide bonds and specific mutations, which improved the antigen stability and immunogenicity of DS-Cav1 (Joyce et al., Nat Struct Mol Biol. (2016) 23(9): 811-820).

[0007] Therefore, there is a need to develop a vaccine that achieves strong protection against RSV by further improving the stability and immunogenicity of the RSV F protein stabilized in a pre-fusion form, such as DS-Cav1 or DS2 variants.

[0008] One example of the present invention provides a recombinant RSV (Respiratory syncytial virus) F protein, comprising an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513 based on the sequence of a precursor polypeptide of a wild-type RSV F protein of SEQ ID NO: 1, but comprising amino acid substitutions S155C, S290C, S190F, V207L, D486L, E487L and F488W.

[0009] Another example of the present invention provides a recombinant RSV F protein comprising an F2 polypeptide of positions 26-103 and an F1 polypeptide of positions 145-513, based on the sequence of a precursor polypeptide of the wild-type RSV F protein of SEQ ID NO: 1, wherein a glycine (Gly)-serine (Ser) peptide linker is introduced between amino acid residues of positions 103 and 145, and comprises amino acid substitutions S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

[0010] Another example of the present invention provides a recombinant RSV F protein further comprising the following amino acid substitutions in the recombinant RSV F protein: (a) A149C and Y458C; (b) N183GC and N428C; or (c) A149C, Y458C, S46G, E92D, S215P, and K465Q.

[0011] Another example of the present invention provides an isolated nucleic acid molecule encoding the recombinant RSV F protein.

[0012] Another example of the present invention provides a vector comprising the nucleic acid molecule.

[0013] Another example of the present invention provides an isolated host cell comprising the nucleic acid molecule.

[0014] Another example of the present invention provides a method for producing a recombinant RSV F protein from an isolated host cell comprising the nucleic acid molecule.

[0015] Another example of the present invention provides an immunogenic composition comprising the recombinant RSV F protein or a nucleic acid molecule encoding the same.

[0016] Figure 1 shows the results of in-silico analyses to evaluate structural stability and interaction changes of RSV fusion proteins in strains (A2 and B18537 strains) containing the DS-Cav1 pre-fusion F protein or the DS-Cav1_SK pre-fusion F protein with an additional substitution from DEF to LLW at amino acid residues 486-488.

[0017] Figure 2 shows the results of in vitro thermostability tests on strains (A2 and B18537 strains) containing the DS-Cav1 pre-fusion F protein or the DS-Cav1_SK pre-fusion F protein with an additional substitution from DEF to LLW at amino acid residues 486-488.

[0018] Figure 3 shows the results of measuring RSV neutralizing antibody titers in serum when strains (A2 and B18537 strains) containing the DS-Cav1 pre-fusion F protein or the DS-Cav1_SK pre-fusion F protein with an additional substitution from DEF to LLW at amino acid residues 486-488 were administered to an animal model.

[0019] Figure 4 shows the results of measuring the pre-fusion F-specific IgG titer and RSV neutralizing antibody titer in serum when strains (A2 and B18537 strains) containing WT, DS-Cav1, DS2-1, DS2-2, DS2-3, or DS2-4 pre-fusion RSV F protein were administered to animal models, respectively.

[0020] Figure 5 shows the results of measuring the pre-fusion F-specific IgG titer and RSV neutralizing antibody titer in serum when strains (A2 and B18537 strains) containing pre-fusion RSV F protein of DS-Cav1, DS2-1, DS2-2, DS2-3 or DS2-4 and strains (A2 and B18537 strains) containing pre-fusion RSV F protein of DS-Cav1_SK DS2-1 SK, DS2-2 SK, DS2-3 SK or DS2-4 SK, which additionally introduced a substitution from DEF to LLW at amino acid residues 486-488, were administered to animal models, respectively.

[0021] Figure 6 shows the results of measuring RSV neutralizing antibody titers in serum when strains (A2 and B18537 strains) containing RSV F protein before DS-Cav1, DS2-1, DS2-2, DS2-3 or DS2-4 were administered alone or in combination at doses of 0.2, 1 or 5 μg to animal models.

[0022] Figure 7 shows the results of measuring pre-fusion F-specific IgG titers and RSV neutralizing antibody titers in serum when various pre-fusion RSV F proteins with or without a substitution of DEF to LLW at amino acid residues 486-488 were administered as mRNA vaccines to animal models.

[0023] According to one aspect of the present invention, a recombinant RSV (Respiratory syncytial virus) F protein is provided, which comprises an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513 based on the sequence of a precursor polypeptide of a wild-type RSV F protein of SEQ ID NO: 1, but comprising amino acid substitutions S155C, S290C, S190F, V207L, D486L, E487L and F488W.

[0024] In one embodiment, the recombinant RSV F protein may comprise an F2 polypeptide of positions 26-103 and an F1 polypeptide of positions 145-513, based on the sequence of the precursor polypeptide of the wild-type RSV F protein of SEQ ID NO: 1, but wherein a glycine (Gly)-serine (Ser) peptide linker is introduced between amino acid residues of positions 103 and 145, and comprises amino acid substitutions S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

[0025] In one embodiment, the recombinant RSV F protein may further comprise the following amino acid substitutions: (a) A149C and Y458C; (b) N183GC and N428C; or (c) A149C, Y458C, S46G, E92D, S215P, and K465Q.

[0026] In one embodiment, the recombinant RSV F protein may have a trimerization domain linked directly or via a linker to the C-terminus. For example, the trimerization domain may be a foldon domain.

[0027] In one embodiment, the recombinant RSV F protein may be derived from an F protein of RSV subtype A, an F protein of RSV subtype B, or a bovine RSV F protein.

[0028] In one embodiment, the recombinant RSV F protein may be a recombinant RSV F protein in which an F2 polypeptide and an F1 polypeptide are linked directly or via a linker.

[0029] In one embodiment, the recombinant RSV F protein may be soluble.

[0030] In one embodiment, the recombinant RSV F protein may exist as a trimer.

[0031] According to another aspect of the present invention, an isolated nucleic acid molecule encoding the recombinant RSV F protein is provided.

[0032] In one embodiment, the nucleic acid molecule may encode a precursor polypeptide of a recombinant RSV F protein.

[0033] In one embodiment, the precursor polypeptide may comprise, in order from N-terminus to C-terminus, a signal peptide, an F2 polypeptide, a p27 polypeptide, an extracellular domain of an F1 polypeptide, and a trimerization domain.

[0034] In one embodiment, the nucleic acid molecule may be an RNA molecule.

[0035] According to another aspect of the present invention, a vector comprising the nucleic acid molecule is provided.

[0036] According to another aspect of the present invention, an isolated host cell comprising the nucleic acid molecule is provided.

[0037] According to another aspect of the present invention, a method for producing a recombinant RSV F protein is provided, comprising the steps of introducing the nucleic acid molecule into an isolated host cell; and culturing the host cell to obtain a recombinant RSV F protein.

[0038] According to another aspect of the present invention, an immunogenic composition comprising the recombinant RSV F protein or a nucleic acid molecule encoding the same is provided.

[0039] In one embodiment, the nucleic acid molecule may encode a precursor polypeptide of a recombinant RSV F protein.

[0040] In one embodiment, the precursor polypeptide may comprise, in order from N-terminus to C-terminus, a signal peptide, an F2 polypeptide, a p27 polypeptide, an extracellular domain of an F1 polypeptide, and a trimerization domain.

[0041] In one embodiment, the nucleic acid molecule may be an RNA molecule.

[0042] In one embodiment, the immunogenic composition may be a vaccine composition.

[0043] Hereinafter, the present invention will be described in more detail.

[0044] Where the terms "comprise," "comprised," or "comprising" are used in this specification (including the claims), they should be construed as specifying the presence of stated features, integers, steps, or components, but not excluding the presence of one or more other features, integers, steps, components, or groups thereof.

[0045] The descriptions of documents, laws, materials, devices, and articles contained herein are included solely to provide context for the present invention. They are not intended to be, in whole or in part, part of the prior art or to suggest or imply that they were common general knowledge in the field to which the present invention pertains prior to the priority date of each claim of this application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that the terms and related definitions used herein are for descriptive purposes only and are not intended to be limiting.

[0047] As used herein, the term “RSV F protein precursor polypeptide” refers to a single 574-amino acid polypeptide initially present for expression of RSV F protein in a cell, also referred to as “F0 polypeptide” or “F0 precursor”. The wild-type precursor polypeptide typically consists of, from N-terminal to C-terminal, a signal peptide (residues 1-25), an F2 polypeptide (residues 26-109), a p27 polypeptide (residues 110-136), and an F1 polypeptide (residues 137-574). During translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum, and the remainder of the F0 precursor (i.e., residues 26-574) is further cleaved by a cellular protease (furin) at two polybasic sites (residues 109 / 110 and 136 / 137), removing the p27 polypeptide (residues 110-136) and forming the RSV F protomer, a heterodimer of F1 and F2 polypeptides linked together via two disulfide bonds. Three protomers assemble to ultimately form the RSV F protein complex (a homotrimer of the three protomers).

[0048] The term “F1 polypeptide (F1)” refers to a polypeptide corresponding to residues 137-574 in the F0 precursor, wherein the wild-type F1 polypeptide is composed of, from N-terminus to C-terminus, an extracellular domain (approximately residues 137-524), a transmembrane domain (approximately residues 525-550), and a cytoplasmic domain (approximately residues 551-574). The F1 polypeptide also contains a hydrophobic fusion peptide at its N-terminus, and also contains two heptad-repeat regions (HRA and HRB), HRA being proximal to the fusion peptide and HRB being proximal to the transmembrane domain.

[0049] The term “F2 polypeptide (F2)” refers to a polypeptide corresponding to residues 26-109 in the F0 precursor.

[0050] The term “soluble F protein” refers to a soluble form of the F protein for use as an RSV vaccine. For example, the soluble F protein may be an F protein lacking part or all of the transmembrane domain and cytoplasmic domain.

[0051] The recombinant RSV F protein herein may be soluble and thus may lack the transmembrane domain and cytoplasmic domain of the wild-type RSV F protein. For example, residues 514 to 574 of the wild-type precursor polypeptide sequence of the RSV F protein may be deleted.

[0052] In a preferred embodiment, the recombinant RSV F protein of the present invention may refer to a soluble F protein stabilized in a pre-fusion conformation. For example, it may refer to a protein comprising at least one epitope specific for the pre-fusion conformation of the RSV F protein, which epitope is determined by specific binding of an antibody specific for the pre-fusion conformation and which can be produced (expressed) in sufficient quantities. An “antibody specific for the pre-fusion conformation” refers to an antibody that specifically binds to the RSV F protein in the pre-fusion conformation but does not bind to the RSV F protein in the post-fusion conformation, and examples thereof include D25, AM22, 5C4, MPE8, and AM14 antibodies.

[0053] For purposes of use as a vaccine, the present invention is characterized by providing a recombinant RSV F protein comprising various modifications to stabilize it in a pre-fusion conformation or to increase antigenic stability and / or immunogenicity.

[0054] One example of the present invention provides a recombinant RSV (Respiratory syncytial virus) F protein, comprising an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513 based on the sequence of a precursor polypeptide of a wild-type RSV F protein of SEQ ID NO: 1, but comprising amino acid substitutions S155C, S290C, S190F, V207L, D486L, E487L and F488W.

[0055] Specifically, substitutions of Ser residues at residues 155 and 290 (S155C and S290C) introduce a disulfide bond, preventing flipping to the post-fusion state, and the co-packing mutations S190F and V207L ​​introduce a larger amino acid to rigidify and stabilize the F protein structure. These RSV F proteins containing the S155C, S290C, S190F, and V207L ​​substitutions can elicit significantly more potent neutralizing antibodies than the post-fusion F protein.

[0056] Additionally, the D486L, E487L, and F488W substitutions structurally stabilize the pre-fusion form of the F protein, and the additional inclusion of the D486L, E487L, and F488W substitutions can increase the structural stability and thermostability of the pre-fusion form of the F protein, thereby increasing neutralizing antibody titers, compared to the case where only the S155C, S290C, S190F, and V207L ​​substitutions are included.

[0057] The present invention also provides a recombinant RSV F protein, comprising an F2 polypeptide of positions 26-103 and an F1 polypeptide of positions 145-513, based on the sequence of a precursor polypeptide of the wild-type RSV F protein of SEQ ID NO: 1, wherein a glycine (Gly)-serine (Ser) peptide linker is introduced between amino acid residues of positions 103 and 145, and comprises amino acid substitutions S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

[0058] Specifically, by further deleting some amino acid residues from the F1 polypeptide and the F2 polypeptide (e.g., further deleting 104-109 from the F2 polypeptide and deleting 137-144 corresponding to the fusion peptide from the F1 polypeptide) and introducing a GS linker between positions 105 and 145, the structural stability can be improved and the neutralizing antibody titer can be increased.

[0059] The present invention also provides a recombinant RSV F protein further comprising the following amino acid substitutions in the recombinant RSV F protein: (a) A149C and Y458C; (b) N183GC and N428C; or (c) A149C, Y458C, S46G, E92D, S215P, and K465Q.

[0060] Specifically, the A149C and Y458C substitutions, and the N183GC and N428C substitutions, respectively, can further increase the physical stability and immunogenicity of the protein by introducing an interprotomer disulfide bond and thereby increasing the interprotomer stability. In addition, the L373R, S46G, E92D, S215P, and K465Q mutations can increase the expression level of the interprotomer variants, increase the physical stability of the pre-fusion form, and further increase immunogenicity.

[0061] The numbering of amino acid residue positions herein is based on the sequence of the wild-type precursor polypeptide of the RSV F protein, e.g., the amino acid sequence of the A2 strain represented by SEQ ID NO: 1. The sequence of SEQ ID NO: 1 contains three naturally occurring substitutions (P102A, I379V, and M447V) in the F0 amino acid sequence of the RSV A2 strain (UniProtKB / Swiss-Prot: P03420).

[0062] Precursor sequence Sequence number Wild type F0MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSY SIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVIITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN1

[0063] The wild-type RSV F protein exhibits remarkable sequence conservation across RSV subtypes. For example, across the F0 precursor molecules, the F proteins of subtypes A and B have approximately 90% amino acid sequence identity, and RSV subtypes A and B each have 81% sequence identity with the bovine RSV F protein. Within RSV subtypes, the F0 sequence identity is even greater; for example, in each of RSV A, B, and bovine subtypes, the RSV F0 precursor proteins have approximately 98% sequence identity. Nearly all identified RSV F0 precursor sequences are 574 amino acids long, typically varying slightly in length due to the length of the C-terminal cytoplasmic tail. In light of this remarkable sequence conservation, one of ordinary skill in the art can readily identify RSV F amino acid positions corresponding to the reference sequence of SEQ ID NO: 1 across different RSV strains. For example, the amino acid positions presented herein can be numbered by reference to the amino acid numbering of SEQ ID NO: 1 by aligning the sequence of another RSV strain with the sequence of SEQ ID NO: 1. It should be noted that different RSV F0 sequences may have different numbering systems, for example, if there are amino acid residues that are added or removed compared to SEQ ID NO: 1. In another embodiment, the recombinant RSV F protein of the present disclosure may have a trimerization domain linked to the C-terminus, either directly or via a linker.

[0064] The term "trimerization domain" refers to a domain capable of promoting trimer formation of F1-F2 heterodimers. Numerous exogenous multimerization domains that promote stable trimer formation of soluble proteins are known in the art. For example, the trimerization domain may be a foldon domain derived from bacteriophage T4 fibritin, consisting of the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 35).

[0065] The foldon domain is located at the C-terminus of the recombinant RSV F protein of the present invention and may be connected directly or via a linker. Examples of the amino acid linker include, but are not limited to, short amino acid linkers such as GG, GS, and SAIG, or longer amino acid linkers containing the repeat sequence GG.

[0066] In another embodiment, the recombinant RSV F protein of the present disclosure may be derived from an F protein of RSV subtype A, an F protein of RSV subtype B, or a bovine RSV F protein. In addition, the recombinant RSV F protein of the present disclosure may be a F2 polypeptide and an F1 polypeptide linked directly or via a linker.

[0067] The present invention also provides an isolated nucleic acid molecule encoding a recombinant RSV F protein.

[0068] The term "nucleic acid," "nucleic acid molecule," or "nucleic acid sequence" refers to a DNA (cDNA or genomic DNA) or RNA molecule or sequence, and "nucleic acid composition" refers to a composition comprising such a nucleic acid, nucleic acid molecule, or nucleic acid sequence. The RNA may be mRNA, viral RNA, self-replicating RNA, circular RNA, or replicon RNA. In a preferred embodiment, the RNA may be mRNA. The RNA, preferably the mRNA, may further comprise at least one selected from a cap structure, a poly(A) sequence, a poly(C) sequence, a histone-stem loop, and / or a 3'-terminal sequence element.

[0069] In one embodiment, the nucleic acid molecule may encode a precursor polypeptide of a recombinant RSV F protein. Furthermore, the precursor polypeptide may comprise, in order from N-terminus to C-terminus, a signal peptide, an F2 polypeptide, a p27 polypeptide, an extracellular domain of an F1 polypeptide, and a trimerization domain.

[0070] Optionally, the precursor polypeptide may further comprise a protease cleavage site and a purification tag at the C-terminus of the trimeric domain. Examples of the protease cleavage site include, but are not limited to, a thrombin cleavage site (LVPRGS) recognized by thrombin. Examples of the purification tag include, but are not limited to, a histidine tag (HHHHHH), a strep tag II (WSHPQFEK), or a c-tag (EPEA).

[0071] As described above, the wild-type precursor polypeptide is typically composed of, from N-terminal to C-terminal, a signal peptide (residues 1-25), an F2 polypeptide (residues 26-109), a p27 polypeptide (residues 110-136), and an F1 polypeptide (residues 137-574). However, during translation, the signal peptide is removed, and the remaining portion of the F0 precursor (i.e., residues 26-574) is further cleaved by a cellular protease (furin) to remove the p27 polypeptide (residues 110-136), resulting in the formation of an RSV F protomer, a heterodimer in which the F1 and F2 polypeptides are linked together via two disulfide bonds, and the resulting recombinant F protein is a trimer in which three protomers are assembled.

[0072] Accordingly, the nucleic acid molecule of the present invention may encode a precursor polypeptide of a recombinant RSV F protein, and the recombinant F protein ultimately expressed therefrom may not include a signal peptide, a p27 polypeptide, a protease cleavage site, a tag, etc. in the composition of the precursor polypeptide described above.

[0073] Nucleic acid molecules can be cloned using conventional molecular biology techniques or created de novo by DNA synthesis using known recombinant techniques.

[0074] The present invention also provides a vector and an isolated host cell comprising the nucleic acid molecule described above.

[0075] To facilitate replication and expression, the nucleic acid molecules of the present invention may be incorporated into a vector, such as an expression vector. The term "vector" refers to a genetic construct containing essential regulatory elements operably linked to allow expression of a genetic insert encoding a protein of interest within the cells of a subject. Various vectors may be used, such as plasmids, viral vectors, bacteriophage vectors, and cosmid vectors. Such vectors can be readily manipulated by methods well known to those skilled in the art, and may be designed to replicate in, for example, prokaryotic and / or eukaryotic cells.

[0076] Host cells for producing recombinant RSV F proteins include prokaryotic (i.e., bacterial) host cells, such as Escherichia coli (E. coli), or eukaryotic host cells. Preferred host cells include, but are not limited to, insect cells such as Spodopterafrugiperda (Sf) cells using the Baculovirus expression system, such as Sf9 and Sf21, Trichoplusiani cells such as Hi-5 cells, and Drosophila S2 cells, and mammalian cells such as Chinese hamster ovary (CHO) cells, such as CHO K1, CHO pro3-, CHO DG44, CHO P12, and the like.

[0077] The present invention also provides a method for producing a recombinant RSV F protein, comprising the steps of introducing the nucleic acid molecule into an isolated host cell; and culturing the host cell to obtain a recombinant RSV F protein.

[0078] Introduction of nucleic acid molecules into cells can be accomplished using any suitable standard technique known in the art, including, but not limited to, transfection, electroporation, electroinjection, microinjection, calcium phosphate co-precipitation, calcium chloride / rubidium chloride, retroviral infection, DEAE-dextran, cationic liposome, polyethylene glycol-mediated uptake, and gene gun.

[0079] Cell culture media are available from a variety of sources, and a suitable medium can typically be selected to allow host cells to express the protein of interest, herein the recombinant RSV F protein. Suitable media may or may not contain serum. Culture conditions, such as temperature, pH, etc., are typically those previously used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0080] The secreted recombinant RSV F protein is recovered from the culture medium. For example, cells can be harvested by deep filtration using first and second filters, centrifugation, etc., disrupted by physical or chemical means, etc., and a crude extract retained for further purification can be generated. Cells used for protein expression can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents, or by other methods well known to those skilled in the art.

[0081] Purification of the recombinant RSV F protein can be achieved through a purification process that includes a chromatographic purification step. Suitable purification methods for the desired protein are known in the art, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion.

[0082] The recombinant RSV F protein provided herein can be used as an immunogenic composition against RSV infection. Accordingly, the present invention also provides an immunogenic composition comprising the recombinant RSV F protein described above, or a nucleic acid molecule encoding the recombinant RSV F protein. The immunogenic composition may be a vaccine composition.

[0083] The term "vaccine" refers to an immunogenic composition designed to reduce or prevent the risk of disease or infection by inducing an immune response, or to improve or treat an existing disease or infection. The vaccine may be multivalent. Vaccines can be used as therapeutic or prophylactic agents.

[0084] The composition of the present invention can simultaneously induce humoral immunity and cellular immunity in an individual. The composition of the present invention can induce antigen-specific T cell responses and / or B cell responses.

[0085] The term "immune response" refers to a change in the activity of cells of the immune system, such as B cells, T cells, or monocytes, as a result of stimulation, either directly or indirectly, through cellular or cytokine mediation. The immune response may be specific (T cell and / or B cell) and / or nonspecific.

[0086] Without limiting the action of the present invention in any way, delivering a composition according to the present invention is useful for inducing an immune response, for example, a T cell response, such as a CD4+ T cell response or a CD8+ T cell response to an antigen, or a B cell response. The CD4+ T cell response and the CD8+ T cell response may occur together with or independently of a humoral response or other specific or nonspecific immune response.

[0087] The compositions of the present invention may be used for the prevention and / or treatment of RSV infection or diseases caused by RSV infection. In certain embodiments, the prevention and / or treatment may target patient groups susceptible to RSV infection. Such target groups include, but are not limited to, elderly individuals (e.g., ≥ 50 years of age, ≥ 60 years of age, and preferably ≥ 65 years of age), adolescents (e.g., ≤ 5 years of age, ≤ 1 year of age), pregnant women (for maternal vaccination), hospitalized patients, and patients who have been treated with antiviral compounds but exhibit an inadequate antiviral response.

[0088] The vaccine composition of the present invention may be a subunit vaccine comprising a recombinant RSV F protein or an RNA vaccine comprising an RNA nucleic acid molecule encoding a recombinant RSV F protein. In a preferred embodiment, the RNA may be mRNA. The RNA, preferably the mRNA, may further comprise at least one selected from a cap structure, a poly(A) sequence, a poly(C) sequence, a histone-stem loop, and / or a 3'-terminal sequence element.

[0089] In one embodiment, the RNA nucleic acid molecule may include a chemical modification.

[0090] As a preferred example, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dehydropseudouridine, 5-methoxyuridine and 2'-O-methyl uridine. There may be more than one type.

[0091] The above chemical modification may occur at the 5-position of uracil. For example, but not limited to, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in an RNA nucleic acid molecule may be replaced with modified uracils (e.g., 5-substituted uracils).

[0092] Other examples of chemical modifications include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-diaza-pseudoisocytidine, 1-methyl-1-diaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-Thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-diaza-adenine, 7-diaza-8-aza-adenine, 7-diaza-2-aminopurine, 7-diaza-8-aza-2-aminopurine, 7-diaza-2,6-diaminopurine, 7-diaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentyladenosine, N6-(cis-hydroxyisopentyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) Adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wiosine, wiobutosine, 7-diaza-guanosine, 7-diaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-diaza-guanosine, 6-thio-7-diaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, Examples include, but are not limited to, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, or N2,N2-dimethyl-6-thio-guanosine.

[0093] In one embodiment, the RNA nucleic acid molecule can be produced via in vitro transcription.

[0094] In vitro transcription of RNA is known in the art (see, e.g., International Publication No. WO2014 / 152027). For example, in vitro transcription requires a linearized DNA template, nucleotide triphosphates, RNA polymerase, a capping enzyme, a ribonuclease inhibitor to prevent RNase contamination, pyrophosphatase to break down pyrophosphate, which inhibits transcription, MgCl2, and an appropriate pH buffer. For example, RNA nucleic acid molecules can be generated in an in vitro transcription reaction using a non-amplified, linearized DNA template. The DNA template can be isolated DNA or cDNA and can include an RNA polymerase promoter, such as a T7 promoter. The RNA polymerase can be a phage RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or a variant thereof. The resulting RNA nucleic acid molecules can be capped via enzymatic capping and purified by chromatographic methods, for example, using an oligo dT substrate.

[0095] In one embodiment, the RNA nucleic acid molecules herein can be formulated in lipid nanoparticles.

[0096] In some embodiments, the lipid nanoparticles can have an average diameter of, but is not limited to, 1 nm or more, 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 6 nm or more, 7 nm or more, 8 nm or more, or 9 nm or more, 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, or 90 nm or more, and 100 nm or less, 200 nm or less, 300 nm or less, 400 nm or less, 500 nm or less, 600 nm or less, 700 nm or less, 800 nm or less, or 900 nm or less.

[0097] Formation of lipid nanoparticles can be accomplished by methods known in the art and / or as described in U.S. Patent Publication No. US 20120178702. In one embodiment, the lipid nanoparticles can comprise a cationic lipid, a non-cationic lipid, a PEG-modified lipid, or a sterol. More specifically, the lipid nanoparticles can comprise, but are not limited to, DLin-MC3-DMA, DLin-DMA, DLin-D-DMA, DLin-K-DMA, C12-200, DLin-KC2-DMA, 98N12-5, DODMA, PLGA, polyethylene glycol (PEG), PEG-DMG, PEG2000-DMG, a pegylated lipid, an amino alcohol lipid, cholesterol, or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0098] As a non-limiting example, the polycation can include a cationic peptide or polypeptide such as, but not limited to, polylysine, polyornithine, and / or polyarginine and a cationic peptide, which are described in International Publication No. WO2012013326 or U.S. Patent Publication No. US 20130142818. In another example, the RNA nucleic acid molecules of the present disclosure can be formulated in lipid nanoparticles comprising a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE). The lipid nanoparticle formulation can be influenced by, but not limited to, the choice of cationic lipid component, the degree of cationic lipid saturation, the nature of the pegylation, the ratios of all components, and biophysical parameters such as size.

[0099] In another embodiment, RNA nucleic acid molecules can also be formulated in liposomes.

[0100] In some embodiments, the liposomes may be, but are not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes, hyaluronan-coated liposomes (Quiet Therapeutics, Israel), or lyophilized gel-phase liposomes (U.S. Patent Publication No. US2012060293).

[0101] The liposome comprises a phospholipid, for example, a phosphatidylcholine which may be non-crystalline at room temperature, such as, but not limited to, egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine, or dilauryl phosphatidylcholine, suitably dioleoylphosphatidylcholine (DOPC). The liposome may also comprise a sterol. Sterols include, but are not limited to, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol, which are well known in the art.

[0102] The compositions of the present invention may further include pharmaceutically acceptable carriers, additives, excipients, diluents, and / or adjuvants. Other examples of carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate, and others are known in the art, for example, Remington's Pharmaceutical Sciences. Adjuvants include (1) aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.), (2) oil-in-water emulsion formulations (with or without specific immunostimulants such as muramyl peptides or bacterial cell wall components), for example, (a) MF59 (WO90 / 14837) comprising 5% squalene, 0.5% Tween 80, and 0.5% Span 85 formulated into submicron particles (optionally, but not necessarily, containing varying amounts of N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), (b) 10%, microfluidized into submicron particles or agitated to produce a large particle size emulsion. SAF comprising squalene, 0.4% Tween 80, 5% pluronic-blocked polymer and N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), and (c) one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), 2% squalene, and 0.RibiTM Adjuvant System (RAS) containing 2% Tween 80; (3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA); (4) Freund's complete adjuvant (CFA) and incomplete adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) ADP-ribosylating toxins of bacteria, such as cholera toxin (CT), pertussis toxin (PT), or heat-labile toxin (LT) of Escherichia coli (E. coli), particularly detoxified mutants of LT-R72, CT-S109, PT-K9 / G129 (WO93 / 13302 and WO92 / 19265); and (7) other substances that act as immunostimulants to enhance the effectiveness of vaccines, including but not limited to.

[0103] The composition can be administered using standard routes of administration. Such routes of administration include, but are not limited to, intramuscular, intranasal, intradermal, subcutaneous, intraarticular, intravenous, intrasplenic, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, sublingual, or intranodal routes. In addition, the composition of the present disclosure can efficiently deliver RNA into the body using, but is not limited to, antigen-presenting cells, a gene gun, or electroporation. The administration regimen can be a single dose or a multiple dose schedule.

[0104] The term "effective amount" refers to an amount sufficient to achieve a desired result when administered to a subject, including a human, e.g., an amount effective in treating or preventing RSV infection. The effective amount can be determined based on the target tissue, target cell type, administration method, size and degree of modification of the RNA nucleic acid molecule, physical characteristics of other components, age, weight, condition, etc. of the subject. The dosage or treatment regimen can be adjusted to provide the optimal therapeutic response, as will be understood by those skilled in the art.

[0105] The present invention will be described in more detail below with reference to the following examples. However, these examples are intended solely to illustrate the invention, and the scope of the invention is not limited by these examples. It will be apparent to those skilled in the art that modifications to the examples described below may be made without departing from the essential spirit of the invention.

[0106]

[0107] summation

[0108] Substituting amino acid residues 486-488 from DEF to LLW can enhance the structural stability of the RSV trimeric fusion protein, referred to as the "SK mutation." This study focused on developing and evaluating stabilized prefusion RSV F proteins, specifically the DS-Cav1 and DS2 variants, by introducing the SK mutation. Both subunit and mRNA vaccine platforms were evaluated in this study. In silico and in vitro assays, the SK mutation significantly improved protein stability and increased neutralizing antibody titers. In immunogenicity tests in animal models, DS-Cav1 SK induced significantly higher neutralizing antibody responses, whereas the DS2 variants, particularly DS2-3 and DS2-4, elicited even higher levels of neutralizing antibodies. Furthermore, while combining different antigen strains can improve cross-reactivity, optimal dosing and combinations are essential for achieving balanced and robust protection against multiple RSV strains.

[0109]

[0110] Example 1. In vitro thermal stability test

[0111] DS-Cav1 and DS-Cav1 SK of strains A2 and B18537 were aliquoted into 50 μL aliquots and heat-treated for 30 minutes in a PCR device. ELISA analysis was performed to analyze the fraction of the pre-trimeric fusion form. The heat-treated samples were diluted to 2 μg / mL with sample coating buffer (1X PBS) onto nickel-coated plates and incubated overnight at 4°C. The captured proteins were sequentially detected with AM14 antibody (anti-F (RSV) AM14 human Fc, 1:2,000) and secondary antibody (goat anti-human IgG Fc cross-conjugated secondary antibody, HRP, 1:10,000). The plates were washed, and 100 μL of TMB substrate was added to each well, incubated at room temperature for 10 minutes, and then placed in the shade. The reaction was terminated by adding 100 μL of TMB stop solution to each well, and the absorbance was measured by selecting two wavelengths of the microplate reader (measurement wavelength: 450 nm, reference wavelength: 650 nm).

[0112]

[0113] Example 2. Immunogenicity study of RSV vaccine candidates

[0114] To evaluate the immunogenicity of RSV vaccine candidates using 6-week-old female BALB / c mice, mice were administered intramuscularly twice at weeks 0 and 3. Serum was collected at week 6 (3 weeks after the last immunization) and neutralizing antibody levels were measured.

[0115] Comparison of immunogenicity of DS-Cav1 and DS-Cav1 SK candidates (Set 1) Group #PlatformStrainAntigen dose (μg / head)AdjuvantMouse #1SubunitA2DS-Cav15AddaVax102DS-Cav1 SK3B18537DS-Cav14DS-Cav1 SK

[0116] Immunogenicity of subunit vaccine candidates (Set 2) Group # Platform Strain Antigen Dose (μg / head) Adjuvant Mouse # 1 Subunit A2DS-Cav 15 AddaVax 10 2DS-Cav 1 SK3DS2-14DS2-1 SK5DS2-26DS2-2 SK7DS2-38DS2-3 SK9DS2-410DS2-4 SK11 B185 37DS-Cav 1 12DS-Cav 1 SK13DS2-114DS2-1 SK15DS2-216DS2-2 SK17DS2-318DS2-3 SK19DS2-420DS2-4 SK

[0117] Evaluation of cross-neutralizing efficacy of monovalent / bivalent subunit vaccine candidates against A2 and B18537 strains (Set 3) Group # Platform Strain Antigen Dose (μg / head) Adjuvant Mouse # 1 Subunit A2 DS2-40.2 AddaVax 102 1354 B18537 0.25 1657 A2 DS2-4 + B18537 DS2-40.1 + 0.18 0.5 + 0.59 2.5 + 2.5

[0118] Immunogenicity of mRNA vaccine candidates (Set 4) Group #PlatformStrainAntigen dose (μg / head)LNP Mouse #1mRNAA2DS-Cav15ALC-0315102DS-Cav1 SK3DS-Cav14DS-Cav1 SK5DS2-16DS2-1 SK7DS2-28DS2-2 SK9DS2-310DS2-3 SK11DS2-412DS2-4 SK13WT14WT SK

[0119] Example 3. Serum binding assay (ELISA)

[0120] Antigen (RSV-preF protein) was diluted to 1 μg / mL using sample coating buffer (1X PBS), dispensed into a 96-well plate, sealed with tape, and incubated overnight at 4°C. The coated plate was washed three times with 300 μL of washing buffer (0.05% Tween-20 in PBS), then blocked with 250 μL of blocking buffer (2% skim milk in PBS) per well, sealed with tape, and incubated for 1 h at RT. Before the blocking step was completed, cluster tubes and sample dilution buffer (2% skim milk and 0.05% Tween-20 in PBS) were prepared to dilute the samples. The samples were serially diluted fivefold starting from 1:50. After the blocking step, the plates were washed, and 50 μL of the diluted serum was dispensed per well into the blocked plates. After dispensing, sealing tape was attached and incubated in a 37°C incubator for 1 hour. 100 μL of diluted secondary antibody (goat anti-mouse IgG, human ads-HRP, 1:2,000) was dispensed into each well. After dispensing, sealing tape was attached, the plate was covered with foil, and incubated in a 37°C incubator for 1 hour. The plate was washed, 100 μL of TMB substrate was added to each well, and incubated at room temperature for 10 minutes and placed in the shade. The reaction was terminated by adding 100 μL of TMB stop solution to each well, and the absorbance was measured by selecting two wavelengths (measurement wavelength: 450 nm, reference wavelength: 650 nm) of a microplate reader.

[0121]

[0122] Example 4. Serum neutralization assay (FRNT)

[0123] 4 x 10 in a 96 deep-well plate one day before virus infection. 4A549 cells / well were prepared. Collected serum was heat-inactivated at 56°C for 30 minutes. Serum samples were serially diluted seven times, mixed with virus (RSV-A2-P5-230724) at a 1:1 ratio, and incubated at 37°C for 1 hour. The initial dilutions of serum and virus were 1 / 10 and 1 / 40 (400 FFU / 100 μL), respectively, in MEM containing 2% FBS. A549 cells were treated with the serum-virus mixture in 96-well plates and incubated at 37°C for 2 hours. The cells were overlaid with 0.8% methyl cellulose in MEM containing 2% FBS and incubated at 37°C for 48 hours. After 48 hours, the cells were fixed with methanol for 24 hours at 4°C. The RSV F protein expressed in infected cells was captured, and then sequentially bound to hRSV-F antibody (1:2,000) and diluted secondary antibody (goat anti-rabbit IgG(H+L)-HRP, 1:5,000). TrueBlue substrate was added to each well and incubated for 20 minutes at room temperature. Foci were counted using a Cytation 7 cell imaging multimode reader. The results were analyzed using a nonlinear regression curve fitting method and the 50% effective concentration (EC 50 ) was calculated.

[0124]

[0125] Experimental results

[0126] 1. Development of a stabilized prefusion RSV F protein through the introduction of SK mutations.

[0127] Substituting amino acid residues 486-488 from DEF to LLW can improve the structural stability of the RSV trimeric fusion protein, a process referred to as the "SK mutation." In this study, we performed in silico analyses to assess the structural stability and interaction changes of the RSV fusion protein following the SK mutation. We also evaluated the in vitro stability and immunogenicity of the protein using an animal immunization model.

[0128]

[0129] 1-1. In silico analysis

[0130] In silico analyses were performed to closely examine the structural properties of the RSV F protein. Homology modeling of DS-Cav1 was performed using the SWISS-MODEL server, using the template structure of bovine RSV F (Protein Data Bank entry, 5TDG). Binding energies and residues were calculated using the MODELLER module of the Discovery Studio 2024 application (BIOVIA, San Diego, CA, USA).

[0131] Substitution of amino acid residues 486–488 from DEF to LLW in the F protein prior to DS-Cav1 fusion results in the formation of a tryptophan cluster at the subunit interface. Furthermore, the leucine substitution at this site reduces negative charge repulsion between subunits of the RSV F trimer. This effect enhances hydrophobic interactions, potentially increasing structural stability and strengthening subunit binding, ultimately leading to the formation of a potentially stabilized trimeric structure (Figure 1).

[0132] In silico analysis revealed that the LLW substitution decreased the potential energy of the DS-Cav1 SK pre-fusion F protein (A2 strain) from -60,601 kcal / mol to -62,971 kcal / mol, indicating a more stable structure. In the B18537 strain, the potential energy slightly increased from -63,324 kcal / mol to -62,855 kcal / mol, suggesting a slight decrease in the stability of this strain. In DS-Cav1 SK, several interacting residues with positions 486-488 were significantly increased. In the A2 strain, the number of interacting residues increased from 24 to 32, and in the B18537 strain, from 24 to 29. This indicates that the LLW substitution enhanced the interactions and strengthened the subunit binding (Table 6).

[0133] In silico analysis of RSV prefusion F protein stability. Strain. RSV F subunit. Potential energy (kcal / mol). Van der Waals energy (kcal / mol). Electrostatic energy (kcal / mol). Number of interacting residues with 446-448. Contact surface area. Number of unfavorable interactions. Strain A2. DS-Cav1-60,601-10,878-56,486. 241,0924. DS-Cav1 SK-62,971-11,468-61,155. 21,1623. B18537. Strain DS-Cav1-63,324-11,913-61,233. 241,0996. DS-Cav1 SK-62,855-11,692-60,956. 291,1964

[0134] 1-2. In vitro thermal stability

[0135] The increased stability observed in silico analyses was confirmed by in vitro thermostability testing. To investigate the thermostability of the engineered pre-fusion RSV F proteins (DS-Cav1 and DS-Cav1 SK), the AM14 (site IV / V) antibody, widely used to measure the trimeric pre-fusion conformation, was used.

[0136] In vitro thermostability tests revealed that substitution of residue 486-LLW-488 significantly increased the melting point of the trimeric structure. While the melting point of DS-Cav1 was 33.14°C, DS-Cav1 SK showed a significantly higher melting point of 64.93°C in the A2 strain. Similarly, the melting point of the B18537 strain increased from 43.22°C to 61.31°C (Fig. 2). These results support the hypothesis that enhanced hydrophobic interactions and tryptophan clustering at the subunit interface contribute to a more thermally stable trimeric structure.

[0137]

[0138] 1-3. Immunogenicity of engineered prefusion RSV F protein

[0139] To evaluate prefusion RSV F proteins with LLW substitutions, the immunogenicity of the DS-Cav1 SK proteins (A2 and B18537) was evaluated in an animal model and compared with that of the DS-Cav1 protein. Mice were immunized intramuscularly with 5 μg of RSV F using AddaVax as an adjuvant at weeks 0 and 3. Animals were bled at week 6, and serum RSV-neutralizing antibody titers were determined.

[0140] The DS-Cav1 SK protein significantly enhanced the ability to induce neutralizing antibodies in mice. This enhancement was observed in both the A2 (7.17-fold) and B18537 (10.61-fold) strains compared to their DS-Cav1 counterparts (Fig. 3).

[0141] In conclusion, substitution of residues 486–488 from DEF to LLW in the pre-DS-Cav1 fusion RSV F protein (DS-Cav1 SK) results in the formation of a hydrophobic cluster, strengthening subunit interactions and enhancing structural stability. This is evidenced by the lower potential energy and greater number of interacting residues in DS-Cav1 SK. The increased stability is further confirmed by the higher melting point observed in thermostability tests, which correlates with significantly higher neutralizing antibody responses in immunized mice. This structural stability leads to enhanced immunogenicity, suggesting that DS-Cav1 SK has the potential to serve as a more stable and immunogenic vaccine candidate against RSV.

[0142]

[0143] 2. Selection of subunit vaccine candidates

[0144] DS-Cav1, a stabilized prefusion form of the fusion glycoprotein, induced a potent protective response against RSV. The DS2 variant possesses genetically linked F subunits, a deleted fusion peptide, and an interprotomeric shift stabilized by an additional disulfide bond and specific mutations. We successfully produced RSV F proteins containing DS-Cav1, the DS2 variant, and the SK mutant (PRT-R03859). The prefusion RSV F protein constructs are presented in Tables 7 and 8. In this study, we evaluated the immunogenicity and protective efficacy of this stabilized F RSV vaccine candidate in an animal immunization model.

[0145]

[0146] 2-1. RSV F vaccine candidate construct

[0147] RSV F vaccine candidate construct list Antigen (A2 strain) Construct sequence number DS-Cav1 DS-Cav1 2 DS-Cav1 SKDS-Cav1 D486L E487L F488W3DS2-1Sc9-10 DS-Cav1 4DS2-1 SKSc9-10 DS-Cav1 D486L E487L F488W5DS2-2Sc9-10 DS-Cav1 A149C Y458C6DS2-2 SKSc9-10 DS-Cav1 A149C Y458C D486L E487L F488W7DS2-3Sc9-10 DS-Cav1 N183GC N428C8DS2-3 SKSc9-10 DS-Cav1 N183GC N428C D486L E487L F488W9DS2-4Sc9-10 DS-Cav1 A149C Y458C S46G E92D S215P K465Q10DS2-4 SKSc9-10 DS-Cav1 A149C Y458C S46G E92D S215P K465Q D486L E487L F488W11

[0148] RSV F vaccine candidate construct list Antigen (B18537 strain) Construct sequence number DS-Cav1 DS-Cav1 12 DS-Cav1 SKDS-Cav1 D486L E487L F488W1 3DS2-1Sc9-10 DS-Cav1 14DS2-1 SKSc9-10 DS-Cav1 D486L E487L F488W1 5DS2-2Sc9-10 DS-Cav1 A149C Y458C 16DS2-2 SKSc9-10 DS-Cav1 A149C Y458C D486L E487L F488W1 7DS2-3Sc9-10 DS-Cav1 N183GC N428C 18DS2-3 SKSc9-10 DS-Cav1 N183GC N428C D486L E487L F488W19DS2-4Sc9-10 DS-Cav1 A149C Y458C S46G E92D S215P K465Q20DS2-4 SKSc9-10 DS-Cav1 A149C Y458C S46G E92D S215P K465Q D486L E487L F488W21

[0149] 2-2. Immunogenicity of pre-fusion RSV F subunit vaccine candidates

[0150] The immunogenicity of pre-fusion RSV F proteins (A2 and B18537) for DS-Cav1, DS2-1, DS2-2, DS2-3, and DS2-4 was evaluated in an animal model. Mice were immunized intramuscularly with 5 μg of RSV F protein using AddaVax as an adjuvant at weeks 0 and 3. At week 6, animals were bled to determine pre-fusion F-specific IgG titers and RSV-neutralizing antibody titers in serum.

[0151] All mice produced antibody titers against the RSV fusion protein as measured by ELISA. The DS2 variants induced significantly higher neutralizing antibody titers against the RSV A2 virus strain compared to the DS-Cav1 and wild-type strains. The significant increases in neutralizing antibody titers observed in the DS2-2 (10.30-fold), DS2-3 (13.36-fold), and DS2-4 (14.34-fold) groups compared to the DS-Cav1 group suggest that these variants have enhanced immunogenic properties. Similarly, for the RSV B18537 virus strain, the DS2-3 (16.32-fold) and DS2-4 (27.46-fold) groups were observed to have significantly increased neutralizing antibody titers compared to the DS-Cav1 group (Fig. 4 and Table 9). Neutralizing antibody responses in both RSV A2 and B18537 virus strains showed that candidates DS2-3 and DS2-4 consistently outperformed the other candidates and were the most effective in inducing neutralizing antibodies.

[0152] Neutralizing antibody titers of RSV F subunit vaccine candidates No. Candidate A2 virus strain GMT (95% CI) B18537 virus strain GMT (95% CI) 1 Wild type (SEQ ID NO: 22) 318.2 (224.8 - 450.5) 373.5 (240.7 - 579.6) 2 DS-Cav 1 370.0 (160.0 - 859.7) 819.4 (338.3 - 1,985) 3 DS2-1 645.4 (307.0 - 1,357) 1,441 (695.3 - 2,987) 4 DS2-2 3,812 (1,998 - 7,272) 6,717 (3,731 - 12,093) 5 DS2-34,946 (2,649 - 9,236)13,364 (7,081 - 25,222)6DS2-45,306 (2,998 - 9,388)22,491 (15,583 - 32,460)

[0153] 2-3. Immunogenicity of RSV F subunit vaccine candidates with SK mutations

[0154] In previous experiments, introduction of the SK mutation into the F protein prior to DS-Cav1 fusion resulted in improved protein structural stability and increased neutralizing antibody titers. To determine whether similar improvements in immunogenicity could be achieved by introducing the SK mutation into DS2 variants, the immunogenicity of DS2-1, DS2-2, DS2-3, and DS2-4 was evaluated by introducing the SK mutation.

[0155] In the case of the A2 virus strain, the introduction of the SK mutation resulted in an approximately 1.69-fold increase in the neutralizing antibody titer of the DS-Cav1 SK group. Furthermore, the neutralizing antibody titers of the DS2-1, DS2-2, and DS2-3 groups increased approximately 2-fold, 1.46-fold, and 1.1-fold, respectively, after the introduction of the SK mutation (Fig. 5 and Table 10).

[0156] For the B18537 virus strain, the DS-Cav1 SK group showed an approximately 4.26-fold increase in neutralizing antibody titer compared to DS-Cav1. In addition, the DS2-1 group showed an approximately 2.4-fold increase in neutralizing antibody titer after the introduction of the SK mutation (Fig. 5 and Table 10).

[0157] These results suggest that introducing the SK mutation into the DS-Cav1 and DS2 variants enhances their immunogenicity. However, while the SK mutation significantly enhanced the immunogenicity of DS-Cav1, the effect on the DS2 variant was more variable, likely due to its inherent structural stability, which may have diminished the impact of the SK mutation. Nevertheless, the DS2 variant vaccines, particularly those introducing the SK mutation, still showed the potential to provide superior protection against RSV, offering a promising avenue for the development of more effective vaccines.

[0158] Neutralizing antibody titers according to SK mutations in the prefusion RSV F protein. No. Group A2 virus strain GMT (95% CI) B18537 virus strain GMT (95% CI) 1DS-Cav1 SK 1,499 (978.6 - 2,296) 1,136 (528.5 - 2,442) 2DS-Cav1 SK 2,533 (1,380 - 4,651) 4,847 (2,474 - 9,495) 3DS2-1 SK 4,54.4 (227.8 - 906.4) 4,19.4 (214.1 - 821.3) 4DS2-1 SK 9,53.1 (443.1 - 2,050) 1,026 (629.9 - 1,673) 5DS2-24,297 (2,289 - 8,064)4,272 (1,931 - 9,451)6DS2-2 SK6,295 (3,860 - 10,265)1,945 (1,151 - 3,285)7DS2-35,062 (2,891 - 8,865)4,440 (2,041 - 9,659)8DS2-3 SK5,554 (3,197 - 9,648)3,925 (1,093 - 14,098)9DS2-45,201 (2,521 - 10,731)21,764 (12,999 - 36,439)10DS2-4 SK3,677 (2,342 - 5,773)20,434 (11,110 - 37,582)

[0159] 3. Neutralizing antibody responses and cross-reactivity of RSV antigens: monovalent and divalent antibody evaluation

[0160] In RSV vaccine development, combining different viral strains is essential to achieve broad protection against diverse viral variants. Monovalent vaccines can provide strong protection against the target strain, but their cross-reactivity with other viral strains is limited. Therefore, to develop more effective vaccines, it is necessary to evaluate how various antigen combinations induce neutralizing antibody responses against various viral strains. To address this need, we conducted experiments to evaluate neutralizing antibody responses using pre-fusion RSV F candidates, including the A2 and B18357 DS2-4 antigens, administered alone or in combination. For the combination groups, equal amounts of each viral strain antigen were mixed to form doses of 0.2, 1, and 5 μg.

[0161] As a result, the A2 DS2-4 antigen alone showed high neutralizing antibody titers against the RSV A2 virus strain, which increased in a dose-dependent manner, reaching a peak titer of 5,306 (95% CI: 2,998 - 9,388) at the 5 μg dose. However, the cross-reactivity against the RSV B18537 virus strain was moderate, with a highest titer of 3,954 (95% CI: 2,411 - 6,485) at 1 μg. When combined with the B18357 DS2-4 antigen, the response against the RSV A2 virus strain was higher than that of B18357 DS2-4 alone, but still lower than that of A2 DS2-4 alone, with a highest titer of 2,190 (95% CI: 1,640 - 2,924) at the 5 μg dose. In contrast, the B18357 DS2-4 antigen alone showed limited cross-reactivity against RSV A2 strains, with low neutralizing antibody titers across all doses, peaking at 543 (95% CI: 72.5 to 1,081) at the 5 μg dose. However, it provided robust protection against the RSV B18537 strain, with a significant and dose-dependent response, reaching a peak titer of 35,833 (95% CI: 23,507 to 54,622) at the 5 μg dose. When combined with the A2 DS2-4 antigen, the response to RSV B18537 virus strain was improved compared to when B18357 DS-24 was administered alone, with a maximum titer of 21,570 (95% CI: 13,403–34,712) at the 5 μg dose, although this was lower than the maximum titer achieved with B18357 DS2-4 alone (Fig. 6 and Table 11).

[0162] In conclusion, the A2 DS2-4 antigen showed high neutralizing antibody titers against RSV A2 strains and moderate cross-reactivity against RSV B18537 strain, whereas the B18357 DS2-4 antigen showed high neutralizing antibody titers against RSV B18537 strain but limited cross-reactivity against RSV A2 strains. Combination of equal amounts of A2 and B18357 DS2-4 antigens improved overall cross-reactivity compared to B18357 DS2-4 alone, but did not exceed the individual potencies of the A2 DS-24 antigen against RSV A2 strains or the B18357 DS2-4 antigen against RSV B18537 strains. These results suggest that antigen combinations can improve responses, but optimizing dose and combination is important to achieve balanced and robust protection against multiple RSV strains.

[0163] Neutralizing antibody titers against RSV A2 and RSV B18537 virus strains: unit price vs. This antibody No. Group (antigen dose, μg) RSV A2 FRNTGMT (95% CI) RSV B18537 FRNTGMT (95% CI) 1A DS2-4 (0.2) 948.0 (557.6 - 1,612) 1,463 (623.6 - 3,432) 2B DS2-4 (0.2) 253.7 (124.8 - 515.6) 7,798 (3,728 - 16,314) 3A DS2-4 (1) 2,891 (1,790 - 4,668) 3,954 (2,411 - 6,485) 4B DS2-4 (1) 466 (271.4 - 800.9) 20,687 (11,191 - 38,238) 5A DS2-4 (5)5,306 (2,998 - 9,388)3,410 (1,862 - 6,246)6B DS2-4 (5)543 (272.5 - 1,081)35,833 (23,507 - 54,622)7A DS2-4 + B DS2-4 (0.2)688.9 (266.0 - 1785)5,829 (3,191 - 10,648)8A DS2-4 + B DS2-4 (1)537.8 (338.1 - 855.6)16,117 (8,287 - 31,345)9A DS2-4 + B DS2-4 (5)2,190 (1,640 - 2,924)21,570 (13,403 - 34,712)

[0164] 4. Selection of mRNA vaccine candidates

[0165] 4-1. Immunogenicity of RSV F mRNA vaccine candidates

[0166] To compare the subunit and mRNA platforms, 12 candidate molecules, including the wild-type, were developed as mRNA vaccines (REP-R02250, REP-R02445) and their immunogenicity was evaluated. While both platforms aim to elicit a strong immune response, mRNA vaccines are particularly characterized by rapid development and strong immunogenicity.

[0167] All tested mRNA vaccines successfully generated robust IgG responses against the RSV F protein. The DS2 variant vaccine demonstrated significantly higher neutralizing antibody levels compared to the other groups, indicating a robust immune response and consistent with the results observed in the subunit platform experiments. The DS2-1, DS2-2, DS2-3, and DS2-4 SK groups all exhibited significantly increased neutralizing antibody titers compared to the DS-Cav1 group (Figure 7).

[0168] The mRNA platform also revealed that the SK mutation significantly impacted neutralizing antibody responses across the various variants, expressed as fold change. For the wild-type RSV F protein (WT), introduction of the SK mutation increased neutralizing antibody titers by approximately 3.6-fold (from 1,202 to 4,308). In the DS-Cav1 group, the SK mutation increased titers by approximately 2.6-fold (from 2,243 to 5,816). This increase was attributed to the enhanced structural and thermostability provided by the SK mutation. However, the effect of the SK mutation was less significant in the DS2 variant, which is a pre-stabilized antigen, resulting in only a modest change in neutralizing antibody levels. These results suggest that the SK mutation significantly increases immunogenicity in the wild-type and DS-Cav1 groups, but has a relatively small effect on the DS2 variant. Nonetheless, DS2 variant vaccines, particularly those introducing the SK mutation, still showed the potential to provide excellent protection against RSV, thus offering a promising avenue for the development of more effective vaccines.

[0169] Neutralizing antibody titers of RSV mRNA vaccine candidates. No. Group sequence number RSV A2 FRNTGMT (95% CI) 1WT 2 3 1,202 (841.7 - 1,715) 2WT SK 2 4 4,308 (2,868 - 6,472) 3DS-Cav 1 2 5 2,243 (987.2 - 5,098) 4DS-Cav 1 SK 2 6 5,816 (3,759 - 8,996) 5DS2-1 2 7 2,663 (1,123 - 6,311) 6DS2-1 SK 2 8 3,299 (2,239 - 4,861) 7DS2-2 2 9 10,566 (7,794 - 14,324) 8DS2-2 SK 3 0 8,207 (4,200 - 16,038)9DS2-3319,669 (5,641 - 16,571)10DS2-3 SK327,883 (4,462 - 13,926)11DS2-43310,010 (7,340 - 13,650)12DS2-4 SK349,805 (5,520 - 17,417)

[0170] 5. Discussion and Conclusion

[0171] This study focused on developing and evaluating prefusion RSV F proteins stabilized by introducing SK mutations, specifically the DS-Cav1 and DS2 variants, and testing them against subunit and mRNA vaccine platforms. The SK mutations were designed to enhance the structural stability and immunogenicity of these RSV trimeric fusion proteins.

[0172] The SK mutation significantly enhanced protein stability by increasing hydrophobic interactions and reducing negative repulsion between subunits. In silico analysis revealed that the DS-Cav1 SK fusion protein had significantly lower potential energy, leading to increased stability. This enhanced stability was experimentally confirmed in vitro by thermal stability tests, where the DS-Cav1 SK mutant exhibited a significantly higher melting point compared to DS-Cav1.

[0173] Immunogenicity assessments in animal models revealed that the DS-Cav1 SK protein significantly increased neutralizing antibody titers compared to DS-Cav1 in both A2 and B18537 virus strains. This suggests that the SK mutation not only structurally stabilizes the protein but also enhances its immunogenic potential. Furthermore, this study evaluated the neutralizing antibody responses and cross-reactivity of various antigen combinations.

[0174] The A2 DS2-4 antigen showed high neutralizing antibody titers against RSV A2 strains, but only moderate cross-reactivity against the B18537 strain.

[0175] In contrast, the B18357 DS2-4 antigen provided strong protection against the B18537 virus strain but showed limited cross-reactivity against the A2 virus strain. When these antigens were combined, overall cross-reactivity was enhanced.

[0176] Furthermore, the evaluation of mRNA vaccine candidates confirmed that all tested mRNA vaccines induced a strong IgG response to the RSV F protein. In particular, the DS2 variant mRNA vaccine elicited significantly higher neutralizing antibody levels, consistent with the results from the subunit vaccine trials.

[0177] Introduction of the SK mutation was effective in enhancing neutralizing antibody responses in the DS2 variant, and was particularly effective in the DS-Cav1 group.

[0178] In conclusion, introduction of SK mutations into DS-Cav1 and DS2 variants enhances structural stability and immunogenicity.

[0179] This study demonstrates that combining different antigenic strains can improve cross-reactivity, but optimal dosing and combinations are essential to achieving balanced and robust protection against multiple RSV strains. These findings provide a strong foundation for the development of more effective RSV vaccines and offer valuable insights for future research and development efforts.

[0180] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical concept or essential characteristics thereof. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. As a recombinant RSV (Respiratory syncytial virus) F protein, Based on the sequence of the precursor polypeptide of the wild-type RSV F protein of SEQ ID NO: 1, comprising an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513, A recombinant RSV F protein comprising amino acid substitutions S155C, S290C, S190F, V207L, D486L, E487L and F488W.

2. In paragraph 1, Based on the sequence of the precursor polypeptide of the wild-type RSV F protein of SEQ ID NO: 1, comprising an F2 polypeptide at positions 26-103 and an F1 polypeptide at positions 145-513, A recombinant RSV F protein comprising a glycine (Gly)-serine (Ser) peptide linker introduced between amino acid residues at positions 103 and 145, and comprising amino acid substitutions S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

3. In paragraph 2, A recombinant RSV F protein further comprising the following amino acid substitutions: (a) A149C and Y458C; (b) N183GC and N428C; or (c) A149C, Y458C, S46G, E92D, S215P, and K465Q.

4. In paragraph 1, The above recombinant RSV F protein is a recombinant RSV F protein in which a trimerization domain is directly connected to the C-terminus or through a linker.

5. In paragraph 4, A recombinant RSV F protein wherein the trimerization domain is a foldon domain.

6. In paragraph 1, The recombinant RSV F protein is a recombinant RSV F protein derived from an F protein of RSV subtype A, an F protein of RSV subtype B, or a bovine RSV F protein.

7. An isolated RNA molecule encoding a recombinant RSV F protein according to any one of claims 1 to 6.

8. In paragraph 7, A nucleic acid molecule, wherein the RNA molecule encodes a precursor polypeptide of a recombinant RSV F protein comprising, from N-terminal to C-terminal, a signal peptide, an F2 polypeptide, a p27 polypeptide, an extracellular domain of an F1 polypeptide, and a trimerization domain.

9. A vector comprising the RNA molecule of clause 7.

10. An isolated host cell comprising the RNA molecule of claim 7.

11. A step of introducing the RNA molecule of clause 7 into an isolated host cell; and Comprising a step of culturing the host cell to obtain a recombinant RSV F protein, A method for producing a recombinant RSV F protein.

12. A recombinant RSV F protein according to any one of claims 1 to 6, or An RNA molecule encoding the recombinant RSV F protein; Immunogenic composition.

13. In paragraph 12, An immunogenic composition, wherein the RNA molecule encodes a precursor polypeptide of a recombinant RSV F protein comprising, from N-terminal to C-terminal, a signal peptide, an F2 polypeptide, a p27 polypeptide, an extracellular domain of an F1 polypeptide, and a trimerization domain.

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