Mutant rsv proteins

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 and a host cell carrying the nucleic acid molecule. The present invention further relates to a method of producing a recombinant RSV F protein. Furthermore, the present invention relates to an immunogenic composition comprising a recombinant RSV F protein or a nucleic acid molecule encoding a recombinant RSV F protein.

Description

Technical Field

[0001] This invention relates to a recombinant RSV (Respiratory Syncytial Virus) F protein, an isolated nucleic acid molecule encoding the recombinant RSV F protein, a vector containing the nucleic acid molecule, and a host cell containing the nucleic acid molecule. Furthermore, this invention relates to a method for producing the recombinant RSV F protein. Additionally, this invention relates to an immunogenic composition comprising the recombinant RSV F protein or a nucleic acid molecule encoding the recombinant RSV F protein. Background Technology

[0002] Respiratory syncytial virus (RSV) is a negative-sense, single-stranded, non-segmented RNA virus belonging to the family Paramyxoviridae. RSV causes lower and upper respiratory tract infections in humans, and although infants and young children are the primary targets of infection, it can also cause fatal respiratory illnesses in immunocompromised patients and the elderly.

[0003] Currently, nirsevimab and palizumab are used as prophylactic monoclonal antibody injections administered to infants and young children to reduce the risk of severe RSV. Arexvy, developed by GSK, is an RSV subunit vaccine for elderly vaccination; Abrysvo, developed by Pfizer, is an RSV subunit vaccine for elderly individuals and pregnant women; and mRESVIA, developed by Moderna, is an RSV mRNA vaccine for elderly vaccination. All three have been approved by the U.S. Food and Drug Administration (FDA). The two aforementioned antibodies target the RSV F protein, while the three vaccines contain a stable pre-fusion recombinant RSV F protein or its mRNA as an antigen.

[0004] RSV's F protein is an abundant fusion glycoprotein in the envelope and exists on the viral surface in a trimer "pre-fusion" state. Once triggered, it inserts a hydrophobic fusion loop into the cell membrane and undergoes extensive conformational changes to fold into an energy-favorable trimer "post-fusion" state, thereby fusing the virus and cell membrane together in the process.

[0005] The RSV F protein is initially expressed as a single 574-residue polypeptide precursor (“F0” or “F0 precursor”) containing a signal peptide sequence at the N-terminus (residues 1-25) and an extracellular domain (ED), a transmembrane domain (TM), and a cytoplasmic tail (CT) comprising three heptapeptide repeat sequences (HRA, HRB, and HRC). Following translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum. The remaining portion of the F0 precursor (i.e., residues 26-574) is further cleaved at two multibase sites (residues 109 / 110 and 136 / 137) by a cellular protease (furin) to remove a 27-amino acid insertion sequence called p27 (residues 110-136), forming the RSV F protomer. The RSV F protomer is a heterodimer in which F1 (residues 137-574) and F2 (residues 26-109) are linked together by two disulfide bonds. The three protomers assemble to form the final RSV F protein complex (a homotrimer of the three protomers).

[0006] In animal models, RSV F proteins stabilized in their pre-fusion form are known to produce stronger neutralizing immune responses than those stabilized in their post-fusion form. For example, “DS-Cav1,” comprising S155C, S290C, S190F, and V207L ​​substitutions, is a recombinant RSV F protein stabilized in its pre-fusion form, and it has been reported to induce neutralizing immune responses that are several times stronger than those observed with the post-fusion form of RSV F protein (McLellan et al., Science (2013) 342, pp. 592-598; International Patent Publication WO2014160463). Furthermore, compared to “DS-Cav1”, the “DS2” variant, as a second-generation antigen, has improved structural stability and immune protective response, with genetically linked F subunits, loss of fusion peptides, and interprotomer movement stabilized by additional disulfide bonds and specific mutations. It has also been reported that it improves the antigenic stability and immunogenicity of DS-Cav1 (Joyce et al., Nat Struct Mol Biol. (2016) 23(9): 811-820). Summary of the Invention

[0007] Technical issues

[0008] Therefore, there is a need to develop a vaccine that provides potent protection against RSV by further improving the stability and immunogenicity of RSV F proteins stabilized in their pre-fusion form (e.g., DS-Cav1 or DS2 variants).

[0009] Technical solutions

[0010] This disclosure provides an example of a recombinant RSV (Respiratory Syncytial Virus) F protein, based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO: 1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513, and amino acid substitutions including S155C, S290C, S190F, V207L, D486L, E487L, and F488W.

[0011] Another example of this disclosure provides a recombinant RSV F protein based on the sequence of the wild-type RSVF protein precursor polypeptide of SEQ ID NO:1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-103 and an F1 polypeptide at positions 145-513, wherein a glycine-serine peptide linker is introduced between amino acid residues at positions 103 and 145, and wherein the recombinant RSV F protein further comprises amino acid substitutions of S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

[0012] Another example of this disclosure provides a recombinant RSV F protein that further comprises the following amino acid substitutions: (a) A149C and Y458C; (b) N183GC and N428C; or (c) A149C, Y458C, S46G, E92D, S215P, and K465Q.

[0013] Another example of this disclosure provides an isolated nucleic acid molecule encoding the recombinant RSV F protein.

[0014] Another example of this disclosure provides a vector containing the nucleic acid molecule.

[0015] Another example of this disclosure provides an isolated host cell containing the nucleic acid molecule.

[0016] Another example of this disclosure provides a method for producing recombinant RSV F protein from isolated host cells containing nucleic acid molecules.

[0017] Another example of this disclosure provides an immunogenic composition comprising recombinant RSV F protein or a nucleic acid molecule encoding recombinant RSV F protein. Attached Figure Description

[0018] Figure 1The results of in-silico computer simulation analysis are shown to assess changes in the structural stability and interactions of RSV fusion proteins in strains (A2 and B18537) containing either the DS-Cav1 pre-fusion F protein or the DS-Cav1_SK pre-fusion F protein, with an additional substitution from DEF to LLW introduced at amino acid residues 486–488.

[0019] Figure 2 Results of in vitro thermostability tests on strains (A2 and B18537) containing either the DS-Cav1 pre-fusion F protein or the DS-Cav1_SK pre-fusion F protein are shown, with an additional substitution from DEF to LLW introduced at amino acid residues 486–488.

[0020] Figure 3 The results show the results of measuring serum RSV neutralizing antibody titers when strains containing DS-Cav1 pre-fusion F protein or DS-Cav1_SK pre-fusion F protein (strains A2 and B18537) were administered to animal models, with an additional substitution from DEF to LLW introduced at amino acid residues 486-488.

[0021] Figure 4 The results show the serum pre-fusion F-specific IgG titer and RSV neutralizing antibody titer when animal models were administered strains (A2 and B18537 strains) containing the pre-fusion RSV F protein of WT, DS-Cav1, DS2-1, DS2-2, DS2-3 or DS2-4, respectively.

[0022] Figure 5 The results show the serum pre-fusion F-specific IgG titer and RSV neutralizing antibody titer when strains containing the pre-fusion RSV F protein of DS-Cav1, DS2-1, DS2-2, DS2-3, or DS2-4 (strains A2 and B18537) and strains containing the pre-fusion RSV F protein of DS-Cav1_SK, DS2-1 SK, DS2-2 SK, DS2-3 SK, or DS2-4 SK (strains A2 and B18537) were administered to animal models, with an additional substitution from DEF to LLW introduced at amino acid residues 486-488.

[0023] Figure 6 The results show the serum RSV neutralizing antibody titers when strains (A2 and B18537 strains) containing the pre-fusion RSV F protein of DS-Cav1, DS2-1, DS2-2, DS2-3 or DS2-4 were administered alone or in combination to animal models at doses of 0.2, 1 or 5 μg.

[0024] Figure 7 The results show the serum titers of pre-fusion F-specific IgG and RSV neutralizing antibodies when various pre-fusion RSV F proteins, with or without the DEF-LLW substitution at amino acid residues 486-488, were administered as mRNA vaccines to animal models. Detailed Implementation

[0025] According to one aspect of the present invention, a recombinant RSV (respiratory syncytial virus) F protein is provided, based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO: 1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513, and comprises amino acid substitutions including S155C, S290C, S190F, V207L, D486L, E487L and F488W.

[0026] In one embodiment, based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO: 1, the recombinant RSV F protein comprises an F2 polypeptide at positions 26-103 and an F1 polypeptide at positions 145-513, wherein a glycine-serine peptide linker is introduced between amino acid residues at positions 103 and 145, and the protein further comprises amino acid substitutions of S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

[0027] 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.

[0028] In one embodiment, the recombinant RSV F protein may have a trimeric domain that is directly or via a linker connected to the C-terminus. For example, the trimeric domain may be a foldon domain.

[0029] In one embodiment, the recombinant RSV F protein may be derived from the RSV A subtype F protein, the RSV B subtype F protein, or the bovine RSV F protein.

[0030] In one embodiment, the recombinant RSV F protein may comprise F2 and F1 peptides, either directly or via a linker.

[0031] In one embodiment, the recombinant RSV F protein may be soluble.

[0032] In one embodiment, the recombinant RSV F protein may exist as a trimer.

[0033] According to another aspect of the invention, an isolated nucleic acid molecule is provided that encodes the recombinant RSV F protein.

[0034] In one embodiment, the nucleic acid molecule may encode a precursor polypeptide of the recombinant RSV F protein.

[0035] In one embodiment, the precursor polypeptide may sequentially include a signal peptide, an F2 peptide, a p27 peptide, an extracellular domain of an F1 peptide, and a trimeric domain from the N-terminus to the C-terminus.

[0036] In one embodiment, the nucleic acid molecule may be an RNA molecule.

[0037] According to another aspect of the present invention, a carrier comprising the nucleic acid molecule is provided.

[0038] According to another aspect of the invention, an isolated host cell containing the nucleic acid molecules is provided.

[0039] According to another aspect of the present invention, a method for producing recombinant RSV F protein is provided, the method comprising introducing the nucleic acid molecule into isolated host cells; and obtaining the recombinant RSV F protein by culturing the host cells.

[0040] According to another aspect of the present invention, an immunogenic composition is provided comprising the recombinant RSV F protein or a nucleic acid molecule encoding the recombinant RSV F protein.

[0041] In one embodiment, the nucleic acid molecule may encode a recombinant precursor polypeptide of the RSV F protein.

[0042] In one embodiment, the precursor polypeptide may sequentially include a signal peptide, an F2 peptide, a p27 peptide, an extracellular domain of an F1 peptide, and a trimeric domain from the N-terminus to the C-terminus.

[0043] In one embodiment, the nucleic acid molecule may be an RNA molecule.

[0044] In one embodiment, the immunogenic composition may be a vaccine composition.

[0045] The invention will be described in more detail below.

[0046] When the terms “comprise”, “comprises”, “comprised” or “comprising” are used in this specification (including the claims), they should be interpreted as specifying the presence of the stated feature, integer, step or component, but do not exclude the presence of one or more other features, integers, steps, components or combinations thereof.

[0047] The descriptions of documents, regulations, materials, devices, articles, etc., in this specification are included only for the purpose of providing context for the invention. They do not imply or suggest that all or part of such descriptions constitute part of the prior art or are common general knowledge in the field prior to the priority date of the claims of this application.

[0048] 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.

[0049] As used herein, the term "RSVF protein precursor polypeptide" refers to the single 574-amino acid polypeptide that is originally present for the expression of RSVF protein in cells, and is also known as the "F0 polypeptide" or "F0 precursor". Wild-type precursor polypeptides typically contain, from N-terminus to C-terminus, 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). Following translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum. The remaining portion of the F0 precursor (i.e., residues 26-574) is further cleaved by a cellular protease (furin) at two multibase sites (residues 109 / 110 and 136 / 137) to remove the p27 polypeptide (residues 110-136), forming the RSVF protomer. The RSVF protomer is a heterodimer in which the F1 and F2 polypeptides are linked together by two disulfide bonds. The three protomers assemble to form the RSVF protein complex (a homotrimer of the three protomers).

[0050] The term "F1 polypeptide (F1)" refers to the polypeptide corresponding to residues 137-574 in the F0 precursor, and the wild-type F1 polypeptide consists of an extracellular domain (approximately residues 137-524), a transmembrane domain (approximately residues 525-550), and a cytoplasmic domain (approximately residues 551-574) from the N-terminus to the C-terminus. Furthermore, the F1 polypeptide contains a hydrophobic fusion peptide (fusion peptide) at the N-terminus and two heptapeptide repeat regions (HRA and HRB), where HRA is near the fusion peptide and HRB is near the transmembrane domain.

[0051] The term "F2 polypeptide (F2)" refers to a polypeptide corresponding to residues 26-109 in the F0 precursor.

[0052] The term "soluble F protein" refers to the soluble form of the F protein used as an RSV vaccine. For example, a soluble F protein may be an F protein in which some or all of its transmembrane and cytoplasmic domains are missing.

[0053] As used herein, recombinant RSV F proteins can be soluble, and therefore the transmembrane and cytoplasmic domains of wild-type RSV F proteins can be deleted. For example, residues from position 514 to 574 can be deleted from the sequence of the wild-type precursor polypeptide of RSV F protein.

[0054] In a preferred embodiment, the recombinant RSV F protein of this disclosure may refer to a soluble F protein stable in its pre-fusion form. For example, it may refer to a protein containing at least one epitope specific to the pre-fusion form of RSV F protein, which is determined by specific binding to an antibody specific to the pre-fusion form and can be produced (expressed) in sufficient quantities. "Antibody specific to the pre-fusion form" refers to an antibody that specifically binds to the pre-fusion form of RSV F protein but not to the post-fusion form of RSV F protein; examples include antibodies D25, AM22, 5C4, MPE8, and AM14.

[0055] For use as a vaccine, the present invention is characterized by providing a recombinant RSV F protein comprising various modifications for a stable pre-fusion form or for increasing antigen stability and / or immunogenicity.

[0056] This disclosure provides an example of a recombinant RSV (Respiratory Syncytial Virus) F protein, based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO:1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513, and amino acid substitutions including S155C, S290C, S190F, V207L, D486L, E487L, and F488W.

[0057] Specifically, by replacing Ser residues (S155C and S290C) with Cys residues at positions 155 and 290, a disulfide bond is introduced to prevent flipping to the post-fusion state. Furthermore, the cavity-filling mutants S190F and V207L ​​enhance and stabilize the F protein structure by introducing bulkier amino acids. This RSV F protein, containing the S155C, S290C, S190F, and V207L ​​substitutions, produces neutralizing antibodies more strongly than the post-fusion F protein.

[0058] Furthermore, the D486L, E487L, and F488W substitutions structurally stabilized the pre-fusion form of the F protein, and compared to the case containing only S155C, S290C, S190F, and V207L ​​substitutions, the additional D486L, E487L, and F488W substitutions increased the structural and thermal stability of the pre-fusion form of the F protein, thereby increasing the neutralizing antibody titer.

[0059] This disclosure also provides a recombinant RSV F protein based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO:1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-103 and an F1 polypeptide at positions 145-513, wherein a glycine-serine peptide linker is introduced between amino acid residues at positions 103 and 145, and the protein further comprises amino acid substitutions of S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

[0060] Specifically, structural stability can be improved and neutralizing antibody titers can be increased by additionally deleting some amino acid residues from the F1 and F2 peptides (e.g., additional deletion of positions 104-109 from the F2 peptide and additional deletion of positions 137-144 corresponding to the fusion peptide from the F1 peptide) and introducing a GS linker between positions 105 and 145.

[0061] This disclosure also provides 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.

[0062] Specifically, A149C and Y458C substitutions, as well as N183GC and N428C substitutions, each introduce interprotomer disulfide bonds to increase interprotomer stability, thereby further enhancing the protein's physical stability and immunogenicity. Furthermore, L373R, S46G, E92D, S215P, and K465Q mutations can increase the expression levels of interprotomer variants, increase the physical stability of the pre-fusion form, and further enhance immunogenicity.

[0063] As used herein, the amino acid residue positions are numbered based on the sequence of the wild-type precursor polypeptide of the RSV F protein, such as the amino acid sequence of strain A2 shown in 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 RSV strain A2 (UniProtKB / Swiss-Prot: P03420).

[0064] [Table 1]

[0065]

[0066] Wild-type RSV F proteins exhibit significant sequence conservation across RSV subtypes. For example, in the F0 precursor molecule, the F proteins of subtypes A and B share approximately 90% amino acid sequence identity, and RSV subtypes A and B each share 81% sequence identity with bovine RSV F proteins. Within RSV subtypes, F0 sequence identity is much greater; for example, the RSV F0 precursor protein shares approximately 98% sequence identity in each of the RSV subtypes A, B, and bovine subtypes. Almost all identified RSV F0 precursor sequences are 574 amino acids in length, typically varying slightly in length due to the length of the C-terminal cytoplasmic tail. Given this significant sequence conservation, even in different RSV strains, those skilled in the art can readily identify the RSV F amino acid positions corresponding to the reference sequence of SEQ ID NO:1. For example, the amino acid positions presented herein can be numbered with reference to the amino acid numbers of SEQ ID NO:1 by comparing the sequences of other RSV strains with the sequence of SEQ ID NO:1. Therefore, it should be noted that different RSV F0 sequences may have different numbering systems if, for example, there are added or removed amino acid residues compared to SEQ ID NO:1.

[0067] In another embodiment, the recombinant RSV F protein of this disclosure may have a trimeric domain that is directly or via a linker connected to the C-terminus.

[0068] The term "trimeric domain" refers to a domain that can promote the formation of trimers from F1-F2 heterodimers. Many exogenous polymerizing domains that promote the formation of stable trimers in soluble proteins are known in the art. For example, a trimeric domain can be a folding domain derived from bacteriophage T4 fibrin, containing the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 35).

[0069] The fold domain is located at the C-terminus of the recombinant RSV F protein of this disclosure and can be directly linked or linked via a linker. Examples of amino acid linkers include, but are not limited to: short amino acid linkers, such as GG, GS, and SAIG; or long amino acid linkers, including the repetitive sequence GG.

[0070] In another embodiment, the recombinant RSV F protein of this disclosure may be derived from the F protein of RSV A isotype, the F protein of RSV B isotype, or bovine RSV F protein. Furthermore, the recombinant RSV F protein of this disclosure may have F2 and F1 peptides directly or via a linker.

[0071] The present invention also provides an isolated nucleic acid molecule encoding a recombinant RSV F protein.

[0072] The terms "nucleic acid," "nucleic acid molecule," or "nucleic acid sequence" refer to DNA (cDNA or genomic DNA) or RNA molecules or sequences, and "nucleic acid composition" refers to a composition comprising such nucleic acids, nucleic acid molecules, or nucleic acid sequences. RNA can be mRNA, viral RNA, self-replicating RNA, circular RNA, or replicon RNA. In a preferred embodiment, the RNA can be mRNA. The RNA, preferably mRNA, may further comprise at least one selected from: cap structures, poly(A) sequences, poly(C) sequences, histone stem-loop structures, and / or 3' end sequence elements.

[0073] In one embodiment, the nucleic acid molecule may encode a recombinant precursor polypeptide of the RSV F protein. Furthermore, the precursor polypeptide may sequentially comprise, from the N-terminus to the C-terminus, a signal peptide, an F2 peptide, a p27 peptide, an extracellular domain of an F1 peptide, and a trimeric domain.

[0074] Optionally, the precursor peptide may also include a protease cleavage site and a tag for purification at the C-terminus of the trimer domain. Examples of protease cleavage sites include, but are not limited to, thrombin-recognized thrombin cleavage sites (LVPRGS). Examples of tags for purification include, but are not limited to, histidine tags (HHHHHH), strep tag II (WSHPQFEK), or c-tag (EPEA).

[0075] As described above, the wild-type precursor polypeptide typically comprises, from N-terminus to C-terminus, 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). Post-translational, 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 RSV F protomers, which are heterodimers in which the F1 and F2 polypeptides are linked together via two disulfide bonds. Finally, the resulting recombinant F protein is a trimer, assembled from three protomers.

[0076] Therefore, the nucleic acid molecule disclosed herein can encode a recombinant precursor polypeptide of RSV F protein, and in the above-mentioned precursor polypeptide configuration, the recombinant F protein ultimately expressed by the recombinant precursor polypeptide may not contain a signal peptide, p27 polypeptide, protease cleavage site, tag, etc.

[0077] This nucleic acid molecule can be cloned using conventional molecular biology techniques, or newly generated through DNA synthesis using known recombination techniques.

[0078] The present invention also provides a vector and an isolated host cell containing the above-mentioned nucleic acid molecules.

[0079] To facilitate replication and expression, the nucleic acid molecules of this disclosure can be integrated into vectors, such as expression vectors. The term "vector" refers to a gene construct containing operatively linked necessary regulatory elements that enable the expression of a gene insert encoding a target protein within the cells of an individual, and various types of vectors can be used, such as plasmids, viral vectors, phage vectors, granular vectors, etc. Such vectors can be readily manipulated by methods well known to those skilled in the art and can be designed to replicate, for example, in prokaryotic and / or eukaryotic cells.

[0080] The host cells used to produce recombinant RSV F protein contain prokaryotic (i.e., bacterial) host cells, such as *Escherichia coli*, or eukaryotic host cells. Preferred host cells are eukaryotic host cells, which may include, but are not limited to, insect cells, such as *Spodoptera frugiperda* (Sf) cells using baculovirus expression systems, such as Sf9 and Sf21; *Trichoplusia ni* cells, such as High Five (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, etc.

[0081] This disclosure also provides a method for generating recombinant RSV F protein, comprising the steps of: introducing nucleic acid molecules into isolated host cells; and obtaining the recombinant RSV F protein by culturing the host cells.

[0082] Nucleic acid molecules can be introduced into cells using appropriate standard techniques known in the art, such as transfection, electroporation, electroinjection, microinjection, calcium phosphate coprecipitation, calcium chloride / rubidium chloride, retroviral infection, DEAE-dextran, cationic liposomes, polyethylene glycol-mediated uptake, gene gun, etc., but not limited to these.

[0083] Cell culture media can be obtained from various sources, and a suitable medium can usually be selected to enable host cells to express the target protein, such as the recombinant RSV F protein in this article. A suitable medium may or may not contain serum. Culture conditions, such as temperature and pH, are those previously used with the host cells selected for expression and are obvious to those skilled in the art.

[0084] Recover the secreted recombinant RSV F protein from the culture medium. For example, cells can be harvested by deep filtration using primary and secondary filters, centrifugation, etc., and disrupted by physical or chemical means to produce a retained crude extract for further purification. Cells used to express the protein can be lysed by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents, or other methods well known to those skilled in the art.

[0085] The recombinant RSV F protein can ultimately be obtained through a purification process that includes chromatographic purification steps. Suitable methods for purifying the target protein are known in the art, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion chromatography.

[0086] The recombinant RSV F protein provided herein can be used as an active ingredient in immunogenic compositions against RSV infection. Therefore, this disclosure also provides an immunogenic composition comprising the recombinant RSV F protein as described above or a nucleic acid molecule encoding the aforementioned recombinant RSV F protein. This immunogenic composition may be a vaccine composition.

[0087] The term "vaccine" refers to an immunogenic composition used to induce an immune response to reduce or prevent the risk of disease or infection, or to improve or treat an existing disease or infection. The vaccine may be multivalent. The vaccine may be used as a therapeutic or preventative agent.

[0088] The compositions disclosed herein can simultaneously induce humoral and cellular immunity in an individual. The compositions disclosed herein can elicit antigen-specific T-cell responses and / or B-cell responses.

[0089] The term "immune response" refers to changes in the activity of cells of the immune system (such as B cells, T cells, or monocytes) as a result of stimulation, directly or indirectly, mediated by cells or cytokines. Immune responses can be specific (T cell and / or B cell) and / or nonspecific.

[0090] Without limiting the scope of the invention in any way, delivery of the compositions according to the invention can be used to induce immune responses, such as inducing T cell responses (e.g., CD4+ T cell responses or CD8+ T cell responses to antigens), or inducing B cell responses. CD4+ T cell responses and CD8+ T cell responses can occur together with humoral responses or other specific or nonspecific immune responses, or independently.

[0091] The compositions disclosed herein can be used for the prevention and / or treatment of RSV infection or disease caused by RSV infection. In some embodiments, prevention and / or treatment may be targeted at patient populations susceptible to RSV infection. Such target populations include, but are not limited to, older adults (e.g., ≥50 years, ≥60 years, and preferably ≥65 years), adolescents (e.g., ≤5 years, ≤1 year), pregnant women (for maternal immunization), hospitalized patients, and patients who have been treated with antiviral compounds but have shown an insufficient antiviral response.

[0092] The vaccine composition disclosed herein may be a subunit vaccine comprising recombinant RSV F protein, or an RNA vaccine comprising an RNA nucleic acid molecule encoding recombinant RSV F protein. In a preferred embodiment, the RNA may be mRNA. The RNA (preferably mRNA) may further comprise at least one selected from: cap structure, poly(A) sequence, poly(C) sequence, histone stem-loop structure, and / or 3' end sequence element.

[0093] In one implementation, the RNA nucleic acid molecule may contain chemical modifications.

[0094] In a preferred embodiment, the chemical modification may be at least one selected from the group consisting of: 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-dihydrouridine, 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, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyluridine.

[0095] Chemical modifications can occur at the 5-position of uracil. For example, 5% or more, 10% or more, 25% or more, 50% or more, 80% or more, 90% or more, or 100% of the uracil in an RNA nucleic acid molecule can be replaced by modified uracil (e.g., 5-position substituted uracil), but are not limited thereto.

[0096] Other examples of chemical modifications may include 5-azacytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudocytidine, pyrrolocytidine, pyrrolo-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopseudocytidine, 4-thio-1-methylpseudocytidine, 4-thio-1-methyl-1-deazapseudocytidine, 1-methyl-1-deazapseudocytidine, zebularine, 5-aza-zebularine, 5-methyl -Zebralin, 5-aza-2-thio-zabralin, 2-thio-zabralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl Adenosine, N6-methyladenosine, N6-isopentyladenosine, N6-(cis-hydroxyisopentyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, inosine, 1-methylinosine, wyosine, wybutosine, 7-deazono-guanosine 7-Deaza-8-aza-guanosine, 6-thioguanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine or N2,N2-dimethyl-6-thio-guanosine, but this disclosure is not limited thereto.

[0097] In one implementation, RNA nucleic acid molecules can be produced through in vitro transcription.

[0098] In vitro transcription of RNA is known in the art (see, for example, International Publication WO2014 / 152027). For example, for in vitro transcription, linearized DNA template, nucleoside triphosphate, RNA polymerase, capping enzyme, ribonuclease inhibitor to prevent RNase contamination, pyrophosphatase to destroy pyrophosphate interfering with transcription, MgCl2, a suitable pH buffer, etc., are required. For example, RNA nucleic acid molecules can be generated in an in vitro transcription reaction using an unamplified linearized DNA template. The DNA template can be isolated DNA or cDNA and may contain an RNA polymerase promoter, such as the T7 promoter. As the RNA polymerase, bacteriophage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or variants thereof, can be used. The resulting RNA nucleic acid molecules can be capped by enzymatic capping and purified by chromatography (e.g., using oligo-dT substrates).

[0099] In one embodiment, the RNA nucleic acid molecules described herein can be formulated in lipid nanoparticles.

[0100] In some embodiments, the average diameter of the lipid nanoparticles can be 1 nm or greater, 2 nm or greater, 3 nm or greater, 4 nm or greater, 5 nm or greater, 6 nm or greater, 7 nm or greater, 8 nm or greater, or 9 nm or greater, 10 nm or greater, 20 nm or greater, 30 nm or greater, 40 nm or greater, 50 nm or greater, 60 nm or greater, 70 nm or greater, 80 nm or greater, or 90 nm or greater, 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, but is not limited thereto.

[0101] The formation of lipid nanoparticles can be achieved by methods known in the art and / or by methods described in US Patent Publication No. US20120178702. In one embodiment, the lipid nanoparticles may comprise cationic lipids, non-cationic lipids, PEG-modified lipids, or sterols. More specifically, the lipid nanoparticles may 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, PEGylated lipids, amino alcohol lipids, cholesterol, or 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC).

[0102] As a non-limiting example, the polycation may comprise cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine, as described in International Publication No. WO2012013326 or US Patent Publication No. US20130142818. In another example, the RNA nucleic acid molecules described herein may be formulated in lipid nanoparticles comprising non-cationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE). Lipid nanoparticle formulations may be influenced by, but are not limited to, the selection of the cationic lipid component, the saturation of the cationic lipid, the PEGylation properties, the ratio of all components, and biophysical parameters (such as size).

[0103] In another implementation, RNA nucleic acid molecules can also be formulated in liposomes.

[0104] In some embodiments, the liposomes may be DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine) liposomes, hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel), or lyophilized gel phase liposomes (US Patent Publication No. US2012060293), but are not limited thereto.

[0105] Liposomes contain phospholipids, such as amorphous phosphatidylcholine at room temperature, such as egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine, or dilauroyl phosphatidylcholine, and suitably, but not limited to, dioleoyl phosphatidylcholine (DOPC). Liposomes may also contain sterols. Sterols are selected from β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol, and are known in the art.

[0106] The compositions described herein may also contain pharmaceutically acceptable carriers, additives, excipients, diluents, and / or adjuvants. Other examples of carriers include colloidal silica, magnesium stearate, cellulose, and sodium lauryl sulfate, as well as other knowledge known in the art, for example, can be found in Remington's Pharmaceutical Sciences. Adjuvants include (1) aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate), and (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), which contains 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-isoglutamyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxyethylamine (MTP-PE)), (b) SAF, comprising 10% squalene, 0.4% Tween 80, 5% Pronic block polymer and N-acetylmurayl-L-threonyl-D-isoglutamine (thr-MDP), microfluidized into submicron particles or stirred to produce large-particle emulsions, and (c) the Ribi™ adjuvant system (RAS) comprising one or more bacterial cell wall components selected from the group consisting of: monophosphoryl lipid A (MPL), trehalose dimethicone ester (TDM) and cell wall skeleton (CWS), 2% squalene and 0.2% Tween 80; (3) a saponin adjuvant, such as Quil A or STIMULON. TM 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) bacterial ADP-ribosylated toxins, such as cholera toxin (CT), pertussis toxin (PT), or heat-labile toxin (LT) of Escherichia coli, especially detoxified mutants of LT-R72, CT-S109, and PT-K9 / G129 (WO93 / 13302 and WO92 / 19265); and (7) other substances that act as immunostimulants to enhance vaccine efficacy, but the composition is not limited thereto.

[0107] This composition can be administered using standard routes of administration. Such routes of administration may include, but are not limited to: intramuscular, intranasal, intradermal, subcutaneous, intra-articular, intravenous, intrasplenic, intrasynovial, intrasternal, intrasheath, intrahepatic, intralesional, intracranial, percutaneous, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, sublingual, or intralymphatic routes. Furthermore, the compositions described herein can be used to efficiently deliver RNA into the body using antigen-presenting cells, gene guns, or electroporation, but are not limited to these methods. Dosing regimens may be single-dose or multiple-dose regimens.

[0108] The term "effective amount" refers to the amount sufficient to achieve the desired effect when applied to an individual (including a human), such as an amount effective in treating or preventing RSV infection. The effective amount can be determined based on the target tissue, target cell type, route of administration, size and degree of modification of the RNA and nucleic acid molecules, physical properties of other components, and the individual's age, weight, and condition. Dosage or treatment regimens can be adjusted to provide the optimal therapeutic response, as understood by one of ordinary skill in the art.

[0109] Embodiments of the present invention

[0110] The invention will be described in more detail below with reference to the following embodiments. However, these are merely illustrative and the scope of the invention is not limited to these embodiments. It will be apparent to those skilled in the art that modifications can be made to the following embodiments without departing from the essential spirit of the invention.

[0111] summary

[0112] The substitution from DEF to LLW improves the structural stability of amino acid residues 486-488 in the RSV trimer fusion protein; this is known as the “SK mutation.” In this study, the invention focuses on developing and evaluating pre-fusion RSV F proteins stabilized by introducing the SK mutation, particularly the DS-Cav1 and DS2 variants. Subunit and mRNA vaccine platforms were evaluated in this study. In computer simulations and in vitro analyses, the SK mutation significantly improved protein stability and increased neutralizing antibody titers. In immunogenicity assays in animal models, the DS-Cav1 SK elicited significantly high neutralizing antibody responses, while the DS2 variants, especially DS2-3 and DS2-4, showed high levels of neutralizing antibodies. Furthermore, although combining multiple antigenic strains can improve cross-reactivity, optimal dosage and combination are crucial for achieving balanced and robust protection against multiple RSV strains.

[0113] Example 1. In vitro thermal stability test

[0114] Take 50 μL of 4 μg / mL DS-Cav1 and DS-Cav1 SK (from strains A2 and B18537) and heat-treat them using a PCR apparatus for 30 minutes. Perform ELISA analysis to determine the proportion of pre-trimeric fusion forms. Dilute the heat-treated samples to 2 μg / mL with sample coating buffer (1X PBS), add them to nickel-coated plates, and incubate overnight at 4°C. Detect captured proteins sequentially with AM14 antibody (anti-F(RSV) AM14 human Fc, 1:2,000) and secondary antibody (goat anti-human IgG Fc cross-adsorption secondary antibody, HRP, 1:10,000). Wash the plates and add 100 μL of TMB substrate to each well, then incubate in the dark at room temperature for 10 minutes. 100 μL of TMB termination solution was added to each well to terminate the reaction, and absorbance was measured using a microplate reader at two wavelengths (measurement wavelength: 450 nm, reference wavelength: 650 nm).

[0115] Example 2. Immunogenicity study of RSV vaccine candidate

[0116] To assess the immunogenicity of the RSV vaccine candidate, 6-week-old female BALB / c mice were administered the candidate intramuscularly twice, at weeks 0 and 3. Serum was collected at week 6 (3 weeks after the last immunization) to measure neutralizing antibody levels.

[0117] [Table 2]

[0118] Comparison of the immunogenicity of DS-Cav1 and DS-Cav1 SK candidates (Group 1)

[0119]

[0120] [Table 3]

[0121] Immunogenicity of subunit vaccine candidates (Group 2)

[0122]

[0123] [Table 4]

[0124] Evaluation of the cross-neutralizing efficacy of monovalent / bivalent subunit vaccine candidates against strains A2 and B18537 (Group 3)

[0125]

[0126] [Table 5]

[0127] Immunogenicity of mRNA vaccine candidates (Group 4)

[0128]

[0129] Example 3. Serum binding assay (ELISA)

[0130] The antigen (RSV-preF protein) was diluted to 1 μg / mL using sample coating buffer (1X PBS), aliquoted into 96-well plates, sealed with sealing tape, and incubated overnight at 4°C. The coated plates were washed three times with 300 μL of wash buffer (0.05% Tween 20 in PBS), then each well was blocked with 250 μL of blocking buffer (2% skim milk in PBS), sealed with sealing tape, and incubated at room temperature for 1 hour. Before the blocking step, cluster tubes and sample dilution buffer (2% skim milk and 0.05% Tween 20 in PBS) were prepared to dilute the samples. Samples were serially diluted 5-fold, starting at 1:50. After the blocking step, the plates were washed, and 50 μL of diluted serum was aliquoted into each well of the blocked plate. After aliquoting, sealing tape was applied, and the plates were incubated at 37°C for 1 hour. Dispense 100 μL of diluted secondary antibody (goat anti-mouse IgG, human ads-HRP, 1:2,000) into each well. After dispensing, apply a sealing tape, cover the plate with foil, and incubate at 37°C for 1 hour. Wash the plate and add 100 μL of TMB substrate to each well, then incubate in the dark at room temperature for 10 minutes. Add 100 μL of TMB stop solution to each well to terminate the reaction, and measure the absorbance at two wavelengths (measurement wavelength: 450 nm; reference wavelength: 650 nm) using a microplate reader.

[0131] Example 4. Serum Neutralization Assay (FRNT)

[0132] One day before viral infection, 4×10⁴ samples were prepared in a 96-well deep plate. 4A549 cells / well. Collected serum was heat-inactivated at 56°C for 30 min. Serum samples were diluted with seven additional 3-fold serial dilutions, mixed with the virus (RSV-A2-P5-230724) at a 1:1 ratio, and incubated at 37°C for 1 h. In MEM containing 2% FBS, the initial dilution factors for serum and virus were 1 / 10 and 1 / 40 (400 FFU / 100 μL), respectively. A549 cells in 96-well plates were treated with the serum-virus mixture and incubated at 37°C for 2 h. Cells were then covered with 0.8% methylcellulose in MEM containing 2% FBS and incubated at 37°C for 48 h. After 48 hours, cells were fixed with methanol at 4°C for 24 h. RSVF protein expressed in infected cells was captured and then sequentially bound with 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 at room temperature for 20 min. Lesions were counted using a Cytation 7 cell imaging multimodal reader. Results were analyzed using nonlinear regression curve fitting and the 50% effective concentration (EC50) was calculated. 50 ).

[0133] Experimental results

[0134] 1. Development of stable pre-fusion RSV F protein by introducing SK mutation

[0135] The substitution of amino acid residues 486-488 from DEF to LLW improves the structural stability of the RSV trimer fusion protein; this modification is termed the "SK mutation." In this study, we performed in-silicoanalysis to assess changes in the structural stability and interactions of the RSV fusion protein following the SK mutation. Furthermore, we evaluated the stability and immunogenicity of the protein in vivo using an animal immunization model.

[0136] 1-1. Computer Simulation Analysis

[0137] Computer simulations were performed to closely examine the structural characterization of the RSV F protein. Homology modeling of DS-Cav1 was performed using the SWIS MODEL server with bovine RSV F (protein database number, 5 TDG) as the template structure. Binding energies and residues were calculated using the MODELLER module of the Discovery Studio 2024 application (BIOVIA, San Diego, CA, USA).

[0138] The substitution of amino acid residues 486-488 from DEF to LLW in the pre-DS-Cav1 fusion F protein resulted in the formation of a tryptophan cluster at the subunit interface. Furthermore, the leucine substitution at this site reduced the negative charge repulsion between the subunits of the RSV F trimer. This effect enhanced hydrophobic interactions, potentially increasing structural stability and strengthening subunit binding, which could ultimately lead to the formation of a potentially stable trimer structure. Figure 1 ).

[0139] Computer simulations showed that LLW substitution reduced the potential energy of the pre-fusion F protein of DS-Cav1 SK (A2 strain) from -60,601 kcal / mol to -62,971 kcal / mol, indicating greater structural stability. In the case of strain B18537, the potential energy slightly increased from -63,324 kcal / mol to -62,855 kcal / mol, indicating a slight decrease in stability. In DS-Cav1SK, the number of interacting residues at positions 486-488 significantly increased. For strain A2, the number of interacting residues increased from 24 to 32, and for strain B18537, it increased from 24 to 29. This indicates that the interactions are strengthened due to LLW substitution, and subunit binding becomes more robust (Table 6).

[0140] [Table 6]

[0141] Computer simulation analysis of F protein stability before RSV fusion

[0142]

[0143] 1-2. In vitro thermal stability

[0144] The increased stability observed in computer simulation analysis was confirmed by in vitro thermostability assays. To investigate the thermostability of engineered pre-fusion RSV F proteins (DS-Cav1 and DS-Cav1 SK), the AM14 (site IV / V) antibody, widely used to measure the pre-fusion form of the trimer, was used.

[0145] As a result of in vitro thermal stability testing, the melting temperature of the trimer structure significantly increased upon substitution of residue 486-LLW-488. The melting temperature of DS-Cav1 was 33.14℃, while DS-Cav1 SK exhibited a significantly higher melting temperature of 64.93℃ in strain A2. Similarly, for strain B18537, the melting temperature increased from 43.22℃ to 61.31℃. Figure 2 These results support the hypothesis that enhanced hydrophobic interactions and tryptophan aggregation at the subunit interface contribute to a more thermally stable trimer structure.

[0146] 1-3. Immunogenicity of RSV F protein before engineered fusion

[0147] To evaluate the pre-fusion RSV F protein with LLW substitution, the immunogenicity of DS-Cav1 SK proteins (A2 and B18537) was assessed in animal models and compared with that of DS-Cav1 protein. Mice were immunized with 5 μg of RSV F intramuscularly at weeks 0 and 3 using AddaVax as an adjuvant. At week 6, blood samples were collected from the animals, and serum RSV neutralizing antibody titers were measured.

[0148] The DS-Cav1 SK protein significantly improved the ability to induce neutralizing antibodies in mice. This improvement was observed in both strains A2 (7.17-fold) and B18537 (10.61-fold) compared to the corresponding DS-Cav1 strains. Figure 3 ).

[0149] In summary, the substitution of residues 486–488 from DEF to LLW in the RSV F protein prior to DS-Cav1 fusion (DS-Cav1SK) leads to the formation of hydrophobic clusters, which enhances inter-subunit interactions and increases structural stability. This is evidenced by the lower potential energy and a greater number of interacting residues in DS-Cav1SK. The increased stability is further confirmed by the higher melting temperature observed in thermal stability tests, which is associated with a significantly higher neutralizing antibody response in immunized mice. This structural stability leads to improved immunogenicity, suggesting that DS-Cav1SK has the potential to serve as a more stable and immunogenic vaccine candidate against RSV.

[0150] 2. Selection of subunit vaccine candidates

[0151] DS-Cav1 comprises a fusion glycoprotein in its stable pre-fusion form and elicits a strong protective response against RSV. The DS2 variant comprises a gene-linked F subunit, a missing fusion peptide, and protomeric motility stabilized by additional disulfide bonds and specific mutations. RSV F proteins including DS-Cav1, the DS2 variant, and the SK mutation (PRT-R03859) were successfully generated. Information on the pre-fusion RSV F protein constructs is provided in Tables 7 and 8. In this study, the immunogenicity and protective efficacy of these stabilized F RSV vaccine candidates were evaluated using animal immunization models.

[0152] 2-1. RSV F vaccine candidate construct

[0153] [Table 7]

[0154] RSV F vaccine candidate construct list

[0155]

[0156] [Table 8]

[0157] RSV F vaccine candidate construct list

[0158]

[0159] 2-2. Immunogenicity of pre-fusion RSV F subunit vaccine candidates

[0160] To assess the immunogenicity of pre-fusion RSV F proteins (A2 and B18537) of DS-Cav1, DS2-1, DS2-2, DS2-3, and DS2-4, evaluations were conducted in animal models. Mice were immunized with 5 μg of RSV F protein intramuscularly at weeks 0 and 3 using AddaVax as an adjuvant. At week 6, blood samples were collected from the animals to determine serum pre-fusion F-specific IgG titers and RSV neutralizing antibody titers.

[0161] All mice produced antibody titers against the RSV fusion protein, as measured by ELISA. The DS2 variant induced significantly higher neutralizing antibody titers against the RSV A2 strain compared to DS-Cav1 and wild-type. Significant increases in neutralizing antibody titers were 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, indicating improved immunogenicity in these variants. Similarly, in the case of the RSV B18537 strain, significantly increased neutralizing antibody titers were observed in the DS2-3 (16.32-fold) and DS2-4 (27.46-fold) groups compared to the DS-Cav1 group. Figure 4 (See Table 9). Neutralizing antibody responses in RSV A2 and B18537 virus strains confirmed that the DS2-3 and DS2-4 candidates consistently performed better than the other candidates and showed the highest efficacy in inducing neutralizing antibodies.

[0162] [Table 9]

[0163] neutralizing antibody titers of RSV F subunit vaccine candidates

[0164]

[0165] 2-3. Immunogenicity of RSV F subunit vaccine candidates with SK mutations

[0166] In previous experiments, introducing the SK mutation into the pre-fusion F protein of DS-Cav1 resulted in improved protein structural stability and increased neutralizing antibody titers. To confirm whether a similar improvement in immunogenicity could be induced by introducing the SK mutation into the DS2 variant, the SK mutation was introduced into DS2-1, DS2-2, DS2-3, and DS2-4 to assess its immunogenicity.

[0167] In the case of the A2 virus strain, the introduction of the SK mutation led to an approximately 1.69-fold increase in neutralizing antibody titers in the DS-Cav1 SK group. Furthermore, after the introduction of the SK mutation, the neutralizing antibody titers in the DS2-1, DS2-2, and DS2-3 groups increased by approximately 2-fold, 1.46-fold, and 1.1-fold, respectively. Figure 5 (and Table 10).

[0168] In the case of the B18537 virus strain, the neutralizing antibody titer in the DS-Cav1 SK group increased by approximately 4.26-fold compared to DS-Cav1. Furthermore, after the introduction of the SK mutation, the neutralizing antibody titer in the DS2-1 group increased by approximately 2.4-fold. Figure 5 (and Table 10).

[0169] These results indicate that the immunogenicity of DS-Cav1 and DS2 variants is improved when the SK mutation is introduced. However, while the SK mutation significantly increases the immunogenicity of DS-Cav1, its effect on the DS2 variant is more variable, possibly because the inherent structural stability of the DS2 variant reduces the impact of the SK mutation. Nevertheless, DS2 variant vaccines, especially those incorporating the SK mutation, still show the potential to provide excellent protection against RSV, potentially offering a promising pathway for developing more effective vaccines.

[0170] [Table 10]

[0171] Neutralizing antibody titers based on the SK mutation in the pre-fusion RSV F protein

[0172]

[0173] 3. Neutralizing antibody response and cross-reactivity to RSV antigen: assessment with monovalent and bivalent antibodies

[0174] In RSV vaccine development, it is necessary to combine various viral strains to achieve broad protection against a wide range of viral variants. Monovalent vaccines can provide strong protection against the target viral strain, but cross-reactivity against other viral strains is limited. Therefore, to develop more effective vaccines, it is necessary to evaluate how various antigen combinations work to elicit neutralizing antibody responses against a variety of viral strains. To address this need, experiments were conducted to evaluate neutralizing antibody responses by administration of pre-fusion RSV F candidates (including A2 and B18357 DS2-4 antigens), alone or in combination. For the combination groups, each viral strain antigen was mixed in equal amounts to constitute doses of 0.2, 1, and 5 μg.

[0175] Therefore, the A2 DS2-4 antigen alone showed high and medium neutralizing antibody titers against RSV A2 strains, which increased in a dose-dependent manner, peaking at 5,306 (95% CI: 2,998–9,388) at a 5 μg dose. However, cross-reactivity against RSV B18537 strains 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 to RSV A2 strains 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 a 5 μg dose. On the other hand, the B18357 DS2-4 antigen alone showed limited cross-reactivity against RSV A2 strains, with low neutralizing antibody titers at all doses, peaking at 543 (295% CI: 72.5–1,081) at a 5 μg dose. However, it provided potent protection against RSV B18537 strains, showing a significant dose-dependent response and reaching a maximum titer of 35,833 (95% CI: 23,507–54,622) at a 5 μg dose. The response against RSV B18537 strains was improved when combined with the A2 DS2-4 antigen compared to B18357 DS-24 alone, with a maximum titer of 21,570 (95% CI: 13,403–34,712) at a 5 μg dose, but this was lower than the peak titer achieved when B18357 DS2-4 was used alone. Figure 6 (and Table 11).

[0176] In summary, the A2 DS2-4 antigen exhibited high and medium neutralizing antibody titers against RSV A2 strains and moderate cross-reactivity against RSV B18537 strains; while the B18537 DS2-4 antigen showed high and medium neutralizing antibody titers against RSV B18537 strains, but limited cross-reactivity against RSV A2 strains. For the combination of equal amounts of A2 and B18537 DS2-4 antigens, the overall cross-reactivity was improved compared to B18537 DS2-4 alone, but did not exceed the individual efficacy of the A2 DS2-4 antigen against RSV A2 strains or the B18537 DS2-4 antigen against RSV B18537 strains. These results indicate that while antigen combinations can improve responses, optimizing dosage and combinations to achieve balanced and robust protection against multiple RSV strains is crucial.

[0177] [Table 11]

[0178] Neutralizing antibody titers against RSV A2 and RSV B18537 strains: monovalent antibodies relative to bivalent antibodies

[0179]

[0180] 4. Selection of mRNA vaccine candidates

[0181] 4-1. Immunogenicity of RSV F mRNA vaccine candidates

[0182] To compare the subunit and mRNA platforms, 12 candidates (including wild-type) were developed as mRNA vaccines (REP-R02250, REP-R02445), and their immunogenicity was evaluated. While both platforms are designed to elicit a strong immune response, the mRNA vaccine platform is characterized by particularly rapid development and strong immunogenicity.

[0183] All tested mRNA vaccines successfully generated a strong IgG response against the RSV F protein. The DS2 variant vaccine showed significantly higher levels of neutralizing antibodies compared to other groups, indicating a strong immune response and consistent with results observed in the subunit platform experiments. The DS2-1, DS2-2, DS2-3, and DS2-4 SK groups all showed significantly increased neutralizing antibody titers compared to the DS-Cav1 group. Figure 7 ).

[0184] The mRNA platform also demonstrated that the SK mutation had a significant impact on neutralizing antibody responses across various variants, expressed as fold changes. In the case of wild-type RSV F protein (WT), the 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 is attributed to the enhanced structural and thermostability provided by the SK mutation. However, in the DS2 variant, which is already a stable antigen, the effect of the SK mutation was less significant, showing only moderate changes in neutralizing antibody levels. These results suggest that while the SK mutation significantly increased immunogenicity in the wild-type and DS-Cav1 groups, its effect on the DS2 variant was relatively small. Nevertheless, DS2 variant vaccines, particularly those with the SK mutation, have demonstrated the potential to provide excellent protection against RSV, thus offering a promising pathway for the development of more effective vaccines.

[0185] [Table 12]

[0186] neutralizing antibody titers of RSV mRNA vaccine candidates

[0187]

[0188] 5. Discussion and Conclusion

[0189] This study focused on the development and evaluation of pre-fusion RSV F proteins, particularly the DS-Cav1 and DS2 variants, which were stabilized by introducing SK mutations; these variants were tested using subunit and mRNA vaccine platforms. The SK mutations were designed to improve the structural stability and immunogenicity of these RSV trimeric fusion proteins.

[0190] The SK mutation significantly increases protein stability by enhancing hydrophobic interactions and reducing negative charge repulsion between subunits. Computer simulations showed that the DS-Cav1 SK fusion-previously-fused F protein exhibited a significantly lower potential energy, indicating increased stability. This improved stability was experimentally confirmed by the DS-Cav1 SK variant exhibiting a significantly higher melting temperature compared to DS-Cav1 in in vitro thermal stability tests.

[0191] As a result of immunogenicity assessment in animal models, the DS-Cav1 SK protein significantly increased neutralizing antibody titers compared to DS-Cav1 in both A2 and B18537 viral strains. This indicates that the SK mutation not only structurally stabilizes the protein but also improves its immunogenic potential. Furthermore, in this study, neutralizing antibody responses and cross-reactivity were evaluated for various antigen combinations.

[0192] The A2 DS2-4 antigen showed high and medium antibody titers against RSV A2 strains, but only moderate cross-reactivity against B18537 strains.

[0193] On the other hand, the B18357 DS2-4 antigen exhibits strong protection against the B18537 virus strain, but shows limited cross-reactivity against the A2 virus strain. When these antigens are combined, the overall cross-reactivity is improved.

[0194] Furthermore, as a result of evaluating mRNA vaccine candidates, all tested mRNA vaccines elicited a strong IgG response against the RSV F protein. In particular, the DS2 variant mRNA vaccine exhibited significantly higher levels of neutralizing antibodies, consistent with the results of subunit vaccine experiments.

[0195] The introduction of the SK mutation is effective in enhancing the neutralizing antibody response in the DS2 variant, and is particularly effective in the DS-Cav1 group.

[0196] In summary, introducing the SK mutation into the DS-Cav1 and DS2 variants improved structural stability and immunogenicity.

[0197] This study demonstrates that while combining multiple antigenic viral strains may improve cross-reactivity, optimal dosage and combination are crucial for achieving balanced and robust protection against multiple RSV viral strains. These findings provide a solid foundation for the development of more effective RSV vaccines and offer valuable insights for future research and development efforts.

[0198] Based on the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. Therefore, it should be understood that the above embodiments are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the following patent claims and their equivalents, rather than the detailed description above.

Claims

1. A recombinant RSV (Respiratory Syncytial Virus) F protein, based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO: 1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-109 and an F1 polypeptide at positions 137-513, and It includes amino acid substitutions of S155C, S290C, S190F, V207L, D486L, E487L and F488W.

2. The recombinant RSV F protein according to claim 1, based on the sequence of the wild-type RSV F protein precursor polypeptide of SEQ ID NO: 1, wherein the recombinant RSV F protein comprises an F2 polypeptide at positions 26-103 and an F1 polypeptide at positions 145-513. in, A glycine-serine peptide linker is introduced between amino acid residues at positions 103 and 145, and contains amino acid substitutions of S155C, S290C, S190F, V207L, L373R, D486L, E487L, and F488W.

3. The recombinant RSV F protein according to claim 2 further comprises the following amino acid substitutions: (a) A149C and Y458C; (b) N183GC and N428C; or (c) A149C, Y458C, S46G, E92D, S215P and K465Q.

4. The recombinant RSV F protein according to claim 1, wherein, The recombinant RSV F protein has a trimeric domain that is directly or via a linker connected to the C-terminus.

5. The recombinant RSV F protein according to claim 4, wherein, The trimeric structural domain is a folded sub-structural domain.

6. The recombinant RSV F protein according to claim 1, wherein, The recombinant RSV F protein is derived from the RSV A subtype F protein, the RSV B subtype F protein, or the bovine RSV F protein.

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

8. The nucleic acid molecule according to claim 7, wherein, The RNA molecule encodes a precursor polypeptide of the recombinant RSV F protein, which, from the N-terminus to the C-terminus, comprises an extracellular domain of a signal peptide, an F2 peptide, a p27 peptide, an F1 peptide, and a trimeric domain.

9. A vector comprising the RNA molecule according to claim 7.

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

11. A method for producing recombinant RSV F protein, the method comprising: Introduce the RNA molecule according to claim 7 into isolated host cells; as well as The recombinant RSV F protein was obtained by culturing the host cells.

12. An immunogenic composition comprising recombinant RSV F protein according to any one of claims 1 to 6, or RNA molecules encoding the recombinant RSV F protein.

13. The immunogenic composition according to claim 12, wherein, The RNA molecule encodes the precursor polypeptide of the recombinant RSV F protein, which, from the N-terminus to the C-terminus, comprises an extracellular domain of a signal peptide, an F2 polypeptide, a p27 polypeptide, an F1 polypeptide, and a trimeric domain.

Images