RSV FG chimeric mRNA vaccine

Abstract

In the present invention, there is discovered a novel mRNA vaccine for preventing respiratory syncytial virus (RSV) infections, the mRNA vaccine being superior in terms of immunogenicity and pharmacological efficacy compared with RSV FG chimeric protein vaccines each comprising RSV F and G proteins and mRNA vaccines which have been produced on the basis of Japanese Patent No. 7253034 (Patent Document 7). In the present invention, an RSV FG chimeric mRNA vaccine is produced by inserting a highly conserved domain of the RSV G protein into a basic skeleton that is formed by linking a transmembrane region to an RSV F protein ectodomain. As a result of performing evaluation on immunogenicity and pharmacological efficacy, it has been confirmed that the immunogenicity and pharmacological efficacy of the RSV FG chimeric mRNA vaccine of the present invention are superior compared with those of the RSV FG chimeric protein vaccines and mRNA vaccines which have been produced on the basis of Patent Document 7.

Description

RSV FG chimeric mRNA vaccine

[0001] RELATED APPLICATIONS This application claims the benefit of priority from Application No. 2024-099798, filed with the Japan Patent Office on June 20, 2024. The priority application is incorporated herein by reference in its entirety.

[0002] The present invention relates to a respiratory syncytial virus (RSV) vaccine. More specifically, the present invention relates to an mRNA encoding a chimeric protein (RSV FG chimeric protein) of the F protein and G protein of respiratory syncytial virus (RSV), which uses a sequence in which a transmembrane domain is fused to the ectodomain of the RSV F protein as a basic backbone, and a portion of the basic backbone is replaced with a portion of the CCD sequence of the RSV G protein, or an mRNA encoding an RSV FG chimeric protein formed by adding a portion of the CCD sequence to the basic backbone.

[0003] Epidemiology and Vaccines: RSV infection is transmitted via droplet or contact, and most people are infected with RSV at least once by the age of two (Non-Patent Document 1). Newborns and the elderly are more likely to experience severe symptoms from RSV infection. In 2019, an estimated 33 million cases of RSV-associated acute lower respiratory tract infection (RSV-ALRI), 3.6 million hospitalizations, 26,300 in-hospital deaths, and 101,400 total deaths were reported worldwide among children aged 0-60 months (Non-Patent Document 2). Among infants aged 0-6 months, an estimated 6.6 million cases of RSV-ALRI, 1.4 million hospitalizations, 13,300 in-hospital deaths, and 45,700 RSV-attributable deaths were reported. The majority of RSV infections among adults are elderly, with an estimated 470,000 hospitalizations and 33,000 in-hospital deaths among adults aged 60 years or older in high-income countries in 2019 (Non-Patent Document 3). To date, recombinant F protein-based vaccines have been approved for RSV. In May 2023, AREXVY (GSK), the world's first RSV vaccine for adults aged 60 years and older, was approved. Furthermore, a Phase 3 clinical trial (NCT04424316) for pregnant women and newborns was conducted, and although not all primary endpoints were achieved, ABRYSVO (Pfizer) was approved in August 2023. ABRYSVO is also approved for elderly patients. Note that AREXVY and ABRYSVO are not intended for pediatric use, and no approved pediatric RSV vaccines exist. As of April 2024, a Phase 3 clinical trial of mRNA-1345 (Moderna), an mRNA vaccine encoding the recombinant F protein, is currently underway. mRNA-1345 is a monovalent mRNA vaccine containing the pre-F antigen derived from the RSV A strain, and therefore tends to have low efficacy against the RSV B strain (Non-Patent Document 4).

[0004] RSV F protein: The RSV F protein is localized on the surface of the RSV envelope and fuses with the host cell membrane to allow the virus to enter host cells, establishing infection. Because RSV F protein exhibits little variation between virus strains, it has been used as a primary vaccine antigen. RSV F protein takes three forms: prefusogenic F, prefusion F (pre-F), and postfusion (post-F) (Patent Documents 1, 2, and 3). The wild-type RSV F protein has a molecular weight of approximately 60 kDa and is composed of domains called signal peptide (SP), fragment 2 (F2), p27, fusion peptide (FP), fragment 1 (F1), transmembrane (TM), and cytoplasmic tail (CT) from the N-terminus. The RSV F protein contains two furin cleavage sites: furin site I cleaves the precursor to prefusogenic F, and furin site II cleaves p27, converting prefusogenic F to pre-F. Pre-F is believed to undergo a structural change to become post-F during membrane fusion between the host cell membrane and the viral envelope. Unlike Post-F, Pre-F is characterized by the presence of a site called site Φ exposed on the molecular surface (Patent Document 3). When wild-type RSV F protein is expressed as a recombinant protein, the above structural change occurs during expression and purification, resulting in the formation of Post-F. Research and development using Post-F and prefusogenic F as immunogens has been conducted in advance, but due to insufficient efficacy, they have not yet been put to practical use (Non-Patent Document 5). For these reasons, a vaccine antigen expected to be more effective than Post-F is considered desirable, and vaccine development using prefusion F as an antigen protein is progressing (Patent Document 1, Non-Patent Document 5).

[0005] RSV G protein: The RSV G protein is localized on the surface of the RSV envelope and binds to host cell receptors, allowing the virus to attach to host cells. The sequence of the RSV G protein varies significantly between virus strains, resulting in subgroups A and B. The G protein of the RSV A2 strain is approximately 30 kDa and consists of, from the N-terminus, the intravirion, transmembrane region (TM), mucin-like region I, conserved central domain (CCD), and mucin-like region II. The CCD region (e.g., amino acid residues 158-199 of SEQ ID NO: 1) is highly conserved among virus strains, with little variation. The chemokine CX3C motif contained in the CCD binds to CX3C receptor 1 (CX3CR1) on the cell surface, promoting virus-cell adhesion and infection (Non-Patent Document 6). The RSV F protein then promotes fusion of the viral envelope with the cell membrane, establishing infection. Since the RSV G protein as well as the RSV F protein is important for infection, vaccine development using the RSV G protein as a vaccine antigen has been carried out (Patent Document 4).

[0006] RSV FG Chimeric Protein: A chimeric respiratory syncytial virus (RSV) polypeptide comprising, from the N-terminus to the C-terminus, (i) a first F protein polypeptide domain, (ii) a G protein polypeptide domain, and (iii) a second F protein polypeptide has been reported as a wild-type RSV F protein and G protein polypeptide, i.e., a wild-type RSV F and G chimeric protein (Patent Document 5). The G protein contained in this chimeric antigen is, for example, any of amino acid residues 183-203, 152-229, 149-229, and 128-229 of SEQ ID NO: 1 (Patent Document 5). In addition, wild-type RSV F and G polypeptides comprising, from the N-terminus to the C-terminus, (i) an amino acid sequence linking a first F protein and a second F protein so as not to be cleaved at the Furin cleavage site, and (ii) a portion of the G protein polypeptide have been reported (Patent Document 6). The G protein contained in this chimeric antigen is, for example, any of amino acid residues 183-203, 152-229, and 149-229 of SEQ ID NO: 1 (Patent Document 6). All of these are RSV FG chimeric proteins. The inventors have previously patented an FG chimeric protein (FΔTM-FG-CCD) in which the ectodomain of the RSV F protein is used as the basic framework (FΔTM) and highly conserved regions of the RSV G protein, excluding the CX3C motif, are inserted or substituted (Patent Document 7). The difference between the present invention and the FG chimera described in Patent Document 7 is that the F protein, which serves as the basic framework, contains not only the ectodomain but also transmembrane domains derived from RSV F and other proteins.

[0007] RSV mRNA vaccines: mRNA vaccines in which mRNA encoding the SARS-CoV-2 S protein antigen is encapsulated in drug delivery systems (DDS) such as lipid nanoparticles (LNPs) or polymers have been reported, and several have already been released on the market. Clinical trial data for COVID-19 vaccines suggest that mRNA vaccine technology has superior efficacy to conventional inactivated and protein-based vaccines, and that it may also be effective in elderly patients (Non-Patent Document 7). Regarding RSV mRNA vaccines, there have been reports of mRNA-based RSV vaccines that encode only the pre-F and post-F ectodomains, or the full length, including the transmembrane domain (Patent Document 8). As shown in the Examples below, an RSV FG chimeric mRNA vaccine (mRNA-LNP FGΔ™) was created and evaluated based on the inventors' prior patent (Patent Document 7). However, its immunogenicity was only comparable to that of a protein-based vaccine (protein FGΔ™). Therefore, an FG chimeric mRNA vaccine (mRNA-LNP FGΔ™) based on the F protein ectodomain as a backbone (mRNA-LNP FGΔ™) was not expected to improve efficacy. When antigen-encoding mRNA is taken up into cells, proteins are synthesized in the cytoplasm. Peptides, which are part of the protein synthesized within the cells, are presented on the cell surface via MHC class I, promoting T cell activation. Furthermore, proteins containing transmembrane domains synthesized within the cells are thought to be expressed on the cell surface and promote B cell activation. Interestingly, mRNA encoding the SARS-CoV S protein to form self-assembling enveloped virus-like particles (eVLPs) not only expresses the S protein on the surface of cells that have taken up the mRNA, but also on the surface of the eVLPs, promoting further B cell activation (Non-Patent Document 23).

[0008] Anti-RSV F antibodies: RSV F protein-specific antibodies are known to have neutralizing activity. There are monoclonal antibodies against multiple epitopes of the RSV F protein (Non-Patent Document 8). One antibody specific to a site called site II of the RSV F protein has been approved as an antibody drug, Palivizumab (Synagis®). This drug has also been approved as a prophylactic drug, but its use is limited to high-risk individuals (premature infants, those with chronic diseases, congenital diseases, etc.) (Non-Patent Document 9). Nisevimab (Beyfortus®), which utilizes half-life extension technology, has been approved as a prophylactic drug for RSV-LRTD in newborns and infants who are born or will experience their first RSV infection during the RSV infection season, and in infants up to 24 months of age who are at high risk of developing severe symptoms from RSV infection during their second RSV infection season.

[0009] Anti-RSV G antibodies: RSV G protein-specific antibodies have been shown to have low neutralizing activity in cells such as A549 cells that do not express CX3CR1. However, when primary human airway epithelial cells that retain CX3CR1 were used, the neutralizing activity of anti-RSV G-CCD antibodies was shown to be equal to or greater than that of anti-RSV pre-F antibodies (Non-Patent Document 10). Several antibodies against the CCD of the RSV G protein are known (Non-Patent Document 11). One of these, the 3D3 antibody, unlike palivizumab, was shown to prevent the exacerbation of symptoms such as airway inflammation caused by infection in a BALB / c mouse model (Patent Document 9, Non-Patent Documents 12 and 13). Furthermore, an antibody called 131-2G has been reported to induce a shift from T helper (Th)2 to Th1 in BALB / c mice during RSV infection (Non-Patent Document 14).

[0010] Among the CCD sequences of the RSV G protein, which are highly conserved among virus strains, there is a particularly highly conserved sequence region, the Central Conserved Region (CCR), located at amino acid residues 164-176 of SEQ ID NO: 1 of the RSV G protein (Non-Patent Documents 15, 16). Monoclonal antibodies that recognize this CCR, 3D3, 131-2G, and 2B11, have been reported to have an inhibitory effect on lung inflammation in a BALB / c mouse model (Non-Patent Document 15). It has been suggested that anti-G antibodies may contribute not only to efficacy but also to safety.

[0011] Mechanism of exacerbation of RSV infection: Clinical trials of a formalin-inactivated RSV vaccine were conducted in the 1960s, but the hospitalization rate after primary RSV infection was 2% in the unvaccinated group and 80% in the vaccinated group, and two children died in the vaccinated group (Non-Patent Document 17). Possible causes of symptom exacerbation due to RSV infection after vaccination include the induction of Th2 immunity by the RSV G protein, the induction of Th2 immunity by carbonylated proteins generated by formalin treatment, and the low avidity of antibodies induced by the vaccine (Non-Patent Documents 18, 19, 20).

[0012] Relationship between RSV G protein and exacerbation mechanism: As mentioned above, infants are known to be particularly susceptible to severe RSV infection. One possible reason for this is a shift in infant immunity to Th2, which increases the number of inflammatory cells. Furthermore, it has been reported in patients with RSV infection that RSV infects neonatal regulatory B lymphocytes (nBregs) via the CX3C motif of the RSV G protein and its receptor CX3CR1, resulting in the production of IL-10, which inhibits the Th1 response and contributes to the exacerbation and severity of symptoms (Non-Patent Document 21).

[0013] Aggravation of symptoms due to low-dose administration: It has been reported that RSV infection after low-dose administration of a vaccine containing the previously developed RSV post F protein (containing a Th1-inducing glucopyranosyl lipid adjuvant-stable emulsion as an adjuvant) and RSV pre F protein (containing a Th2-inducing aluminum hydroxide hydrate gel as an adjuvant) resulted in vaccine-enhanced disease such as alveolitis in a cotton rat model (Non-patent document 22).

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[0016] Clinical trial data for mRNA vaccines against SARS-CoV-2 have shown that mRNA vaccines are more effective than conventional inactivated and protein-based vaccines, and are also sufficiently effective in the elderly. An mRNA vaccine encoding only the RSV F protein is currently under development for RSV. Based on a prior patent (Patent Document 7) for a protein vaccine using the FG chimeric protein discovered by the inventors as an antigen, an RSV FG chimeric mRNA vaccine (FΔTM-FG mRNA) was created and evaluated, using the ectodomain of the F protein excluding the transmembrane region as the base structure. The vaccine was found to have the same immunogenicity as a protein-based vaccine (FΔTM-FG protein), indicating that switching to the mRNA modality alone would not be expected to improve efficacy.

[0017] The present inventors have created a new RSV FG chimeric mRNA vaccine based on a prior patent (Patent Document 7) in order to solve the above problems, that is, to improve immunogenicity and efficacy. Specifically, an RSV FG chimeric mRNA vaccine with enhanced immunogenicity and efficacy was created by inserting a highly conserved domain of the RSV G protein into a sequence in which the RSV F protein ectodomain is linked to a transmembrane domain as a basic framework. According to the present invention, by creating an FG chimeric mRNA in which the transmembrane domain is fused to the RSV F protein ectodomain, significant improvements in immunogenicity and efficacy were observed compared to protein-based vaccines.

[0018] Previous vaccines that use the RSV pre-F protein as a vaccine antigen induce anti-RSV F antibodies that are protective against infection, but do not induce anti-RSV G antibodies that are protective against infection.

[0019] By administering an mRNA vaccine encoding an RSV FG chimeric protein, anti-RSV G antibodies are induced in addition to anti-RSV F antibodies. The difference from the prior patent (Patent Document 7) is that the RSV F protein ectodomain contains a transmembrane region.

[0020] This results in an immune induction that is different from that of protein vaccines or mRNA vaccines that do not contain transmembrane domains, enhancing immunogenicity, i.e., improving the ability to induce anti-RSV F antibodies and anti-RSV G antibodies, suggesting that these antibodies are the main drivers of improved efficacy.

[0021] The present invention provides the following [1] to

[24] . [1] An mRNA encoding a chimeric protein (RSV FG chimeric protein) of the F protein and G protein of respiratory syncytial virus (RSV), wherein the RSV FG chimeric protein is formed by substituting a portion of the CCD sequence of the RSV G protein or adding a portion of the CCD sequence to the basic backbone, using a sequence in which the RSV F protein ectodomain is fused with a transmembrane domain. [2] The mRNA according to [1], wherein a portion of the CCD sequence encodes a sequence in which multiple sequences excluding the CX3C motif of the RSV G protein sequence of SEQ ID NO: 1 are linked together. [3] The mRNA according to [1], wherein a portion of the CCD sequence encodes a sequence in which two or three sequences from positions 162 to 171 and positions 187 to 197 of the RSV G protein of SEQ ID NO: 1 are linked together. [4] The mRNA according to [1], wherein a portion of the CCD sequence encodes a sequence in which two or three sequences from positions 162 to 172 and 187 to 199 of the RSV G protein of SEQ ID NO: 1 are linked together. [5] The mRNA according to [1], wherein a portion of the CCD sequence encodes a sequence in which a sequence excluding the CX3C motif of the RSV G protein sequence of SEQ ID NO: 1 is linked with a linker. [6] The mRNA according to any one of [1 to 5], wherein the linker linking multiple sequences excluding the CX3C motif is GGGGS (SEQ ID NO: 3) or EAAAK (SEQ ID NO: 4). [7] The mRNA according to [1], wherein a portion of the FP domain of the RSV F protein is replaced with a portion of the CCD sequence according to any one of [2 to 6]. [8] The sequence corresponding to positions 137-146 of the RSV F protein set forth in SEQ ID NO: 2 is replaced with a portion of the CCD sequence of any of [2-6], or the sequence corresponding to positions 137-146 of the RSV F protein is omitted, and a portion of the CCD sequence encoding a sequence in which multiple sequences excluding the CX3C motif in the RSV G protein sequence of SEQ ID NO: 1 are linked is added. [9] The mRNA of [1], wherein a portion of the CCD sequence described in [2-6] is added to the N-terminus or C-terminus of the transmembrane region fused to the RSV F protein ectodomain.

[10] The mRNA of [1], wherein a portion of the CCD sequence of any of [2-6] is added between the p27 and FP domains of the RSV F protein.

[11] The mRNA of [1], wherein a portion of the CCD sequence of any of [2-6] is added to the C-terminus of the F2 domain of the RSV F protein.

[12] The mRNA of any of [1-10], wherein a portion of the CCD sequence of any of [2-6] is linked to the RSV F protein or transmembrane region via a linker.

[13] The mRNA of any of [1-12], wherein the linker linking the region in which the transmembrane region is fused to the ectodomain of the RSV F protein to the backbone and the portion of the CCD sequence is GGGGS (SEQ ID NO: 3) or EAAAK (SEQ ID NO: 4).

[14] The mRNA of any of [1-13], wherein the ectodomain of the RSV F protein encodes an amino acid sequence having 90% or more sequence identity to the amino acid sequence of positions 1-513 of SEQ ID NO: 2.

[15] The mRNA of any one of [1-14], wherein the ectodomain of the RSV F protein encodes an amino acid sequence comprising a pre-F mutation.

[16] The mRNA of any one of [1-15], wherein the transmembrane region encodes an amino acid sequence derived from a viral protein.

[17] The mRNA of any one of [1-16], wherein the transmembrane region encodes an amino acid sequence derived from the RSV F protein.

[18] The mRNA of any one of [1-16], wherein the transmembrane region encodes an amino acid sequence derived from an influenza virus HA protein.

[19] The mRNA of

[18] , wherein the influenza virus HA protein is an H1N1, H2N2, or H3N2 HA protein.

[20] The mRNA of any one of [1-15], wherein the transmembrane region encodes an amino acid sequence derived from a human protein.

[21] The mRNA according to

[20] , wherein the human protein is selected from the group consisting of growth hormone receptor, low-density lipoprotein receptor, glycophorin-B, beta-1,3-galactosyltransferase 1, beta-1,3-galactosyltransferase 4, linker for activation of T-cells family member 1, lymphocyte transmembrane adapter 1, linker for activation of T-cells family member 2, and phosphoprotein associated with glycosphingolipid-enriched microdomains 1.

[22] The mRNA according to any one of [1 to 21], wherein the transmembrane region has a hydrophobic amino acid ratio of 70% or more.

[23] The mRNA according to any one of [1 to 22], wherein the transmembrane region has a hydrophobic amino acid ratio of 80% or more.

[24] The mRNA according to any one of [1 to 23], wherein the transmembrane region has a hydrophobic amino acid ratio of 90% or more.

[25] The mRNA of any of [1 to 24], wherein the F protein has a mutation corresponding to S155C, S290C, S190F, or V207L ​​in SEQ ID NO: 2, or a mutation corresponding to T103C, I148C, S190I, and D486S in SEQ ID NO: 2.

[26] DNA for synthesizing the mRNA of any of [1 to 25].

[27] A protein comprising the amino acid sequence of any of [1 to 25].

[28] A RSV vaccine comprising the mRNA of any of [1 to 25].

[0022] According to the present invention, it is possible to provide a vaccine that is effective in preventing RSV infection.

[0023] Schematic diagram of the RSV FG chimeric protein sequence. Immunogenicity evaluation of RSV FG chimeric mRNA vaccine: anti-F antibody titer (B6 mice). Immunogenicity evaluation of RSV FG chimeric mRNA vaccine: anti-F antibody titer (BALB / c mice). Immunogenicity evaluation of RSV FG chimeric mRNA vaccine: anti-G antibody titer (B6 mice). Immunogenicity evaluation of RSV FG chimeric mRNA vaccine: anti-G antibody titer (BALB / c mice). Efficacy evaluation of RSV FG chimeric mRNA vaccine: neutralizing antibody titer (B6 mice). Efficacy evaluation of RSV FG chimeric mRNA vaccine: neutralizing antibody titer (BALB / c mice). Comparison by mouse age - immunogenicity evaluation: anti-F antibody titer (B6 mice). Immunogenicity evaluation 2 of RSV FG chimeric mRNA vaccine: anti-F antibody titer (BALB / c mice). Immunogenicity evaluation 2 of RSV FG chimeric mRNA vaccine: anti-G antibody titer (BALB / c mice). Efficacy evaluation 2 of RSV FG chimeric mRNA vaccine: neutralizing antibody titer (BALB / c mice). Evaluation of FG chimeric protein expression in HEK293 cells: flow cytometry. Evaluation 2 of FG chimeric protein expression in HEK293 cells: flow cytometry. Immunogenicity evaluation 3 of RSV FG chimeric mRNA vaccine: anti-F antibody titer (BALB / c mice). Efficacy evaluation 3 of RSV FG chimeric mRNA vaccine: neutralizing antibody titer (BALB / c mice). Immunogenicity evaluation 4 of RSV FG chimeric mRNA vaccine: anti-F antibody titer (BALB / c mice). Immunogenicity evaluation 4 of RSV FG chimeric mRNA vaccine: anti-G antibody titer (BALB / c mice). Immunogenicity evaluation 4 of RSV FG chimeric mRNA vaccine; Neutralizing antibody titer (BALB / c mice). Efficacy evaluation 4 of RSV FG chimeric mRNA vaccine; Neutralizing antibody titer against RSV B strain (BALB / c mice). Efficacy evaluation 4 of RSV FG chimeric mRNA vaccine; Protective ability against mouse infection - virus copy number in lung tissue (BALB / c mice). Efficacy evaluation 4 of RSV FG chimeric mRNA vaccine; Ratio of protected mice to the number of infected individuals (BALB / c mice). Immunogenicity evaluation 5 of RSV FG chimeric mRNA vaccine; Anti-F antibody titer (BALB / c mice). Immunogenicity evaluation 5 of RSV FG chimeric mRNA vaccine; Anti-G antibody titer (BALB / c mice). Efficacy evaluation 5 of RSV FG chimeric mRNA vaccine; Neutralizing antibody titer (BALB / c mice).

[0024] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0025] (RSV FG chimeric mRNA vaccine) The vaccine (RSV FG chimeric mRNA vaccine) of this embodiment is an mRNA encoding a chimeric protein (RSV FG chimeric protein) of the RSV F protein and G protein, and is a basic structure in which a sequence in which the RSV F protein ectodomain is fused with a transmembrane domain is used as a basic structure, and a portion of the basic structure is replaced with a portion of the CCD sequence of the RSV G protein, or a portion of the CCD sequence is added to the basic structure. It is a formulation based on an mRNA encoding an RSV FG chimeric protein. Here, "addition" includes the meaning of "insertion", and "substitution" or "addition" may be via a linker between the basic structure. Examples of amino acid sequences of linkers include GGGGS (SEQ ID NO: 3) or EAAAK (SEQ ID NO: 4). In addition, "part" is defined as a sequence containing at least 5 amino acids. "mRNA" may be a self-amplifying self-amplifying RNA (saRNA) or a trans-amplifying RNA (taRNA).

[0026] The ectodomain of the RSV F protein refers to the portion of the RSV F protein that is exposed to the outside of the cell membrane. The ectodomain of the RSV F protein is, for example, positions 1 to 513 of SEQ ID NO: 2 or a sequence corresponding thereto. The ectodomain of the RSV F protein may include positions 514 to 529 of SEQ ID NO: 2 or a sequence corresponding thereto, excluding positions 530 to 550 of SEQ ID NO: 2, which is considered to be the transmembrane domain (TM domain), or a sequence corresponding thereto. A portion of the RSV G protein may include, for example, a sequence of 10 or more amino acids in the highly conserved region of positions 158 to 199 of SEQ ID NO: 1 or a sequence corresponding thereto, particularly a highly conserved sequence region, the central conserved region (CCR), of positions 164 to 176 of SEQ ID NO: 1 or a sequence corresponding thereto, preferably excluding positions 182 to 186 of SEQ ID NO: 1, which is the CX3C motif, or a sequence corresponding thereto. In addition, multiple portions of the G protein contained in the FG chimeric protein may be linked, and linkers such as GGGGS (SEQ ID NO: 3) or EAAAK (SEQ ID NO: 4) may be used for linking. When multiple portions of the CCD sequence are added or substituted, the added or substituted portions of the CCD sequence may be the same or different CCD sequence portions. When multiple portions of the CCD sequence are added or substituted, the added or substituted CCD sequence may be 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 3 to 5, and most preferably 3, but is not limited thereto. In addition, the addition or substitution of portions of the CCD sequence may be used in combination. In a preferred embodiment, a portion of the CCD sequence is added. In the present disclosure, from the viewpoint of preventing symptom exacerbation and aggravation, it is preferred that the portion of the CCD sequence added or substituted does not contain a CX3C motif. In one embodiment, the sequence corresponding to positions 137 to 146 of the RSV F protein set forth in SEQ ID NO: 2 may be deleted. In another embodiment, the sequence corresponding to positions 137-146 of the RSV F protein set forth in SEQ ID NO: 2 may be replaced with a portion of the CCD sequence.

[0027] The transmembrane domain (TMD) is a sequence that penetrates a lipid membrane or a viral envelope, and its amino acid residues are often hydrophobic. The transmembrane domain fused to the RSV F protein ectodomain may be an amino acid sequence that penetrates or anchors the cell membrane, such as a viral protein, a human protein, or a sequence containing hydrophobic amino acids. Specifically, the transmembrane domain may be derived from the RSV F protein, RSV G protein, or RSV SH protein, or from an influenza virus HA protein, NA protein, or SARS-CoV-2 S protein. Examples of influenza virus HA proteins include the TMDs of the HA proteins of influenza viruses H1N1, H2N2, and H3N2. When derived from a human protein, the transmembrane domain may be type I, type II, type III, or type IV. Furthermore, the transmembrane domain may be derived from a protein other than a human protein, or may be an artificially modified human protein. Examples of human-derived TMDs include, but are not limited to, Type I: Growth hormone receptor_P10912, Low-density lipoprotein receptor_P01130, Glycophorin-B_P06028; Type II: Beta-1,3-galactosyltransferase 4_O96024, Beta-1,3-galactosyltransferase 1_Q9Y5Z6; Type III: Linker for activation of T-cells family member 1_O43561, Lymphocyte transmembrane adapter 1_Q8IWV1, Linker for activation of T-cells family member 2_Q9GZY6, Phosphoprotein associated with glycosphingolipid-enriched microdomains 1_Q9NWQ8.The transmembrane region may contain hydrophobic amino acids at a ratio of 60% or more, 70% or more, 80% or more, 90% or more, or 100%. If the purpose is to express a target protein on the cell membrane, the transmembrane region may preferably contain 70% or more, more preferably 80% or more, 90% or more, or 100%. The sequence of the transmembrane region may include the cytoplasmic region of the F protein. Preferably, the sequence may be positions 551 to 574 of SEQ ID NO: 2, or a part thereof.

[0028] The substitution or addition position of a portion of the G protein may be any position in the sequence in which the transmembrane domain is fused to the ectodomain of the RSV F protein. Specifically, it may be the C-terminus of the F2 domain, the N-terminus of the FP domain, the C-terminus of the ectodomain of the RSV F protein, or the C-terminus of the transmembrane domain. A portion of the G protein may be substituted or added between the p27 and FP domains of the RSV F protein. Preferably, the F protein is located around position 109, around position 137, around position 513, or a sequence corresponding thereto in SEQ ID NO: 2.

[0029] Proteins containing transmembrane domains may be difficult to secrete into cell culture supernatants due to the presence of a transmembrane region. This difficulty in secretion may make it difficult to obtain the protein. In the present disclosure, the transmembrane region is fused to the ectodomain of the RSV F protein in the mRNA encoding the RSV FG chimeric protein, which may be advantageous in terms of obtaining vaccine components compared to using a chimeric protein containing a transmembrane domain as a vaccine. In one embodiment of the present disclosure, mRNAs in which the transmembrane region is fused to the ectodomain of the RSV F protein may have enhanced infection protection capabilities compared to mRNAs without a transmembrane region fused thereto.

[0030] The sequence of the RSV F protein that becomes part of the RSV FG chimeric protein can be a sequence derived from a known wild-type RSV strain or a clinical isolate. For example, it can be a sequence registered in the GenBank, European Bioinformatics Institute (EBI), and DNA Data Bank of Japan (DDBJ) databases. A representative sequence of the RSV F protein is the sequence set forth in SEQ ID NO: 2. The RSV F protein may include, from the N-terminus, a region called an SP domain, an F2 domain, a p27 domain, an FP domain, and an F1 domain. The RSV F protein may further include, from the N-terminus, a region called a TM domain and a CT domain after the F1 domain. In the present invention, the CT domain may be included as part of the FG chimeric protein. The F protein may include a mutation associated with a DS-Cav1 mutation that maintains a stable preF structure, and these mutations may be S155C, S290C, S190F, or V207L. The F protein may include the amino acid mutations T103C, I148C, S190I, and D486S.

[0031] The sequence of the RSV G protein that forms part of the RSV FG chimeric protein can be a sequence derived from a known wild-type RSV strain or a clinical isolate. For example, it can be a sequence registered in the GenBank, European Bioinformatics Institute (EBI), and DNA Data Bank of Japan (DDBJ) databases. A representative sequence of the RSV G protein is SEQ ID NO: 1 (Human respiratory syncytial virus A2, UniProtKB / Swiss-Prot: Accession No. P03423). A representative sequence of the G protein CCD of the RSV B strain is SEQ ID NO: 5 (Human respiratory syncytial virus B (strain B1), UniProtKB / Swiss-Prot: Accession No. O36633).

[0032] Although the CCD sequence of the G protein is conserved among virus strains, the portion of the CCD sequence excluding the CCR sequence has about 75% sequence identity to SEQ ID NO: 1 even among known wild-type strains and clinical isolates. In the present invention, a portion of the CCD sequence may have 75% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity to SEQ ID NO: 1. Note that, since the sequence of the F protein differs from SEQ ID NO: 2 by about 90% depending on the known wild-type strain, the F protein may have preferably 90% or more, more preferably 95% or more, and more preferably 99% or more sequence identity to SEQ ID NO: 2.

[0033] In the present disclosure, a "corresponding sequence" may refer to a sequence that has 85%, 90%, 95, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100% sequence identity to a parent sequence. Where sequence identity is less than 100%, for example, the "corresponding sequence" may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide or amino acid substitutions, deletions, additions, or combinations thereof, relative to the parent sequence.

[0034] The RSV FG chimeric mRNA vaccine has superior infection protection ability compared to conventional protein-based vaccines. To examine the infection protection ability, neutralizing antibody titer assessment, viral copy number after virus challenge, or viral titer may be measured.

[0035] The mRNA vaccine of the present invention may be administered as mRNA alone, or may be administered using a drug delivery system (DDS), such as a lipid nanoparticle (LNP), polymeric micelle, or liposome.

[0036] LNPs may generally be composed of four types of lipids: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-modified lipids. The lipid ratios constituting the LNP may be 40-70% ionizable lipids, 1-10% phospholipids, 20-50% cholesterol, and 0.1%-5% PEG-modified lipids.

[0037] The ionizable lipid may be a cationic lipid or an anionic lipid, and specific cationic lipids may be D-Lin-MC3-DMA, DOTAP, DODAP, DODMA, CL1, L319, ALC-315, SM-102, LPO1, Lipid5, Lipid8, Lipid29, TCL-053, C12-200, cKK-E12, CL4H6, YSK05, YSK13, YSK15, COATSOME SS-E, COATSOME SS-EC, COATSOME SS-OC, COATSOME SS-OP, CL4F, CL6F, CL15F, CL16F, CL17F, CL4F, CL15F, etc.

[0038] Polymeric micelles may be formed from cationic block copolymers composed of PEGylated polyamines and mRNA. Specific block copolymers may be PEG-PAsp(DET), PEG-PAsp(TET), or PEG-PAsp(TEP).

[0039] The RSV FG chimeric mRNA vaccine may contain an additive, such as an amino acid, a sugar, a surfactant, or a combination thereof.

[0040] The dosage form of the RSV FG chimeric mRNA vaccine may be, for example, but is not limited to, a liquid, a powder (lyophilized powder, dry powder), a capsule, a tablet, or a frozen state.

[0041] The RSV FG chimeric mRNA vaccine can also include a pharmaceutically acceptable carrier, such as saline, buffered saline, dextrose, water, glycerol, an isotonic aqueous buffer, an emulsifier, a pH adjuster, or a combination thereof.

[0042] The RSV FG chimeric mRNA vaccine may be administered by syringe, transdermal patch, microneedle, implantable sustained release device, syringe with microneedle, needle-free device, nasal spray, orally, or sublingually.

[0043] Mammals that can be vaccinated with the RSV FG chimeric mRNA vaccine include, for example, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, monkeys, humans, etc. The RSV F / G chimeric vaccine of the present invention is most preferably used in humans, and can be used in both men and women aged 5 to 64, as well as pregnant women, infants, children under 5 years of age, and adults aged 65 years and older.

[0044] The number of administrations of the RSV FG chimeric vaccine of the present invention varies depending on the purpose of administration, the administration method, and the condition of the recipient (gender, age, weight, medical condition), but when administered to humans, it may be administered once, twice, or three times.

[0045] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0046] Materials and Methods for the Examples 1. Plasmid DNA Construction The coding sequence (CDS) of the RSV FG chimeric protein was artificially synthesized (Eurofins Genomics) and amplified by PCR. A template plasmid for in vitro transcription (IVT) was constructed using the Cloning Kit for mRNA Template (Takara, product code 6143). The CDS contained 15-base overlap regions with both ends of the Linearized Template Vector included in the kit. DNA was prepared by In-Fusion cloning according to the kit's instructions. The Linearized Template Vector contained a T7 promoter, the transcription initiation sequence AGG, the 5'-UTR, the 3'-UTR, and a PolyA sequence (105 bases). After In-Fusion cloning, minipreps were performed using a QIAprep Spin Miniprep Kit (QIAGEN), and the target sequence was confirmed by sequence analysis. Subsequently, midiprep was performed using NucleoBond Xtra Midi Plus (MACHEREY-NAGEL), and the target sequence was confirmed by sequence analysis.

[0047] 2. mRNA Preparation. The prepared plasmid DNA was linearized by Hind III restriction enzyme digestion, and mRNA was synthesized by in vitro transcription (IVT) using the IVTpro T7 mRNA Synthesis Kit (Takara, product code 6144) and CleanCap Reagent AG (TriLink). IVT was performed according to the kit's instructions, except that N1-methylpseudouridine (m1Ψ) was used instead of UTP. Following the IVT reaction, template DNA was removed with DNase I and mRNA was purified with lithium chloride (LiCl), according to the kit's instructions. The prepared mRNA was transfected into HEK293FT cells using the RiboJuice mRNA Transfection Kit (Merck, product code TR-1013), and expression of the target protein was confirmed by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. The expression of the target protein in the culture supernatant was confirmed by the ELISA method described in the prior patent (Patent Document 7), and the expression of the target protein on the cell membrane was confirmed by flow cytometry using an anti-RSV F antibody (anti-site II antibody) and an anti-RSV G antibody (3D3 antibody).

[0048] 3. LNP mRNA was prepared in 25 mM citrate buffer (pH 4.86), and lipids were dissolved in ethanol at the lipid component ratio specified in the package insert for Comirnaty Intramuscular Injection (Pfizer). The mRNA aqueous solution and lipid ethanol solution were filtered through a 0.22 μm filter. Using a syringe pump (YMC, product code YSP-201) and the iLNP1.0 microchannel (Lilac Pharma), the above mRNA aqueous solution and lipid ethanol solution were mixed at a flow rate ratio of mRNA:lipid = 3:1 and collected in an RNase-free tube. The resulting mRNA-LNP was dialyzed against a sucrose solution containing the components specified in the package insert for Comirnaty Intramuscular Injection (Pfizer) and concentrated using an Amicon Ultra-100K (MERCK). The particle size of the mRNA-LNP was measured using a Zetazizer Nano or Zetazizer Ultla (Malvern Panalytical) and confirmed to be approximately 70-110 nm. The percentage of mRNA encapsulated in LNPs. （商標） The RiboGreen RNA Assay Kit (Thermo) was used to confirm an encapsulation rate of approximately 90%. Expression of the target protein on the cell membrane of the prepared mRNA-LNP was confirmed by flow cytometry using anti-RSV F antibody (anti-site II antibody) and anti-RSV G antibody (3D3 antibody).

[0049] 4. Immunogenicity Test: C57BL / 6J mice (Jackson Laboratory Japan, female, 5 weeks old), BALB / c mice (Japan SLC, female, 5 weeks old), and C57BL / 6J-Aged mice (Jackson Laboratory Japan, female, 81 weeks old) were acclimated for several days to a week before use. The sample was inoculated into the thighs (50 μL per foot, totaling 100 μL) via syringe. Two inoculations were administered at 3-week intervals, and blood was collected 3 weeks after the second inoculation. Blood was collected in Separapid Microtubes S (FUCHIGAMI), left to stand for at least 30 minutes, and then centrifuged to separate the serum. Anti-F antibody titers in the serum were measured using the following ELISA method. Anti-F antibody titer ELISA: RSV F protein was immobilized on a 96-well Pierce Nickel-Coated Plate (Thermo Fisher Scientific). After washing, 1% Bovine Serum Albumin-Phoshate Buffer Saline (BSA-PBS) was applied and allowed to stand for 1 hour. After washing, anti-mouse IgG HRP-labeled antibody was applied, sealed, and allowed to stand for 1 hour. After washing, 3,3',5,5'-Tetramethylbenzidine Liquid Substrate (Sigma-Aldrich) was applied and allowed to stand for 30 minutes. After stopping color development with 1N H2SO4, absorbance was measured using a SPECTRAMAX 190 (Thermo). For a standard calibration curve, the concentration of anti-F antibody was converted using a dilution series of anti-site II antibody.

[0050] The anti-G antibody titer in the serum obtained above was measured using the following ELISA method. Anti-G antibody titer ELISA: A peptide with a cysteine ​​added to the C-terminus of the sequence linking positions 158-172 and 187-199 of SEQ ID NO: 1 of the G protein was synthesized (Eurofins Genomics) and immobilized on Pierce Maleimide Activated Plates (Thermo, product code 15153). Excess maleimide groups were inactivated with 10 μg / mL L-cysteine ​​hydrochloride, and then a sample diluted with 1% BSA-PBS was applied. After incubation for 1 hour, HRP-Rabbit Anti-Mouse IgG (H+L) (Life Technologies) was applied and incubated for 1 hour. A 50:50:1 mixture of QuantaRed Enhancer Solution, QuantaRed Stable Peroxide, and QuantaRed ADHP Concentrate was added to the reaction mixture. After 1-15 minutes of incubation, the plate was shaken for 10-30 seconds and the reaction was stopped with Stop Solution. Absorbance (excitation 530 nm / absorption 590 nm) was measured using a SYNERGY HT fluorescence microplate reader (BioTeK). A standard calibration curve was prepared by converting the anti-G antibody concentration to a dilution series of the 3D3 antibody.

[0051] 5. Drug efficacy evaluation: Neutralizing antibody titer and protective effect against infection in mice. The neutralizing antibody titer in the serum obtained above was measured by the following method. Hep-2 cells were cultured at 2 × 10 5Cells were seeded at 1000 cells / mL into a 96-well plate and cultured for 1 day at 37°C under 5% CO2. RSV dilutions were prepared using medium containing 1 / 50 of the rabbit serum complement (Cedarlane). Equal volumes of serum diluted in medium and RSV dilutions were mixed and incubated at 37°C under 5% CO2 for 1 hour. The serum-RSV reaction mixture was added and incubated at 37°C under 5% CO2 for 1 hour. The serum-RSV reaction mixture was then removed, the plate was washed with PBS, and medium without complement was added. The plate was then cultured at 37°C under 5% CO2 for 3–5 days. After removing the reaction mixture, the plate was washed with PBS, methanol was added, and the plate was incubated at room temperature for 30 minutes. After removing the methanol and air-drying, the plate was washed with PBS, and anti-RSV F antibody dilutions were applied and incubated at 37°C for 1 hour. After removing the anti-RSV F antibody dilutions, anti-mouse IgG Alexa488-labeled antibody (Abcam) dilutions were applied, and the plate was incubated at 37°C for 1 hour. After removing the diluted anti-mouse IgG Alexa488-labeled antibody (Abcam), the plate was washed with PBS. Diluted Hoechst 33342 solution (Dojindo Laboratories) was applied and left to stand in the dark for 10 minutes. The nuclear stain was removed, and the plate was washed with PBS. PBS was then applied to the plate and analyzed using an image analyzer. Based on the infection rate of the serum dilution series and the infection rate of the RSV-only wells, curve fitting was performed using GraphPad Prism 9 (GraphPad Software) according to the fitting guide, and the neutralizing antibody titer (IC50) was calculated.

[0052] The mouse protection assessment method was as follows. BALB / c mice were inoculated twice with 5 μg / dose of mRNA-LNP at 3-week intervals, followed by intranasal challenge with RSV A2 (ATCC) 3 weeks later. The whole lungs of the mice were removed, and viral RNA was extracted by disrupting the lung tissue using TRIzol (Thermo Fisher Scientific), Lysing Matrix D (MP-Bio), and FastPrep-24 (MP-Bio). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and viral copy number was calculated by qPCR. qPCR conditions were as follows: Pretreatment was performed using a mixture of a sense primer (CARCAAAGTTAYTCTATCATGTC (SEQ ID NO: 6)), an antisense primer (GATCCTGCATTRTCACARTACCA (SEQ ID NO: 7)), a minor groove binder (MGB) probe (TGTAGTACAATTRCCACT (SEQ ID NO: 8); except that the 3' end of the standard DNA was modified with a labeled fluorescent dye and the 3' end was modified with MGB and Eclipse Quencher [MGBEQ]), and distilled water (NIPPON GENE). 10 2 ~10 7A standard curve was created using a range of copies. Real-time PCR was performed at 50°C for 2 minutes, followed by 95°C for 10 minutes, followed by 50 cycles of 95°C for 30 seconds and 60°C for 1 minute. A standard curve was created from the amplification curve of the standard material, and the viral copy number of the sample was calculated using QuantStudio 7 (Applied Biosystems). Samples below the detection limit were analyzed with a lower limit of 100 copies.

[0053] The amino acid sequence for the mRNA-encoded RSV FG chimeric protein is shown in Table 1 below.

[0054]

[0055] Example 1 An mRNA-LNP containing an mRNA encoding an RSV FG chimeric protein was prepared (FIG. 1). Specifically, using the 1-513 positions of the RSV F full length (RSV F (full)) excluding the TM domain as the basic backbone, the FP domain positions 137-146 of the F protein were replaced with positions 162-171 of the G protein (mRNA-LNP FGΔTM001), mRNA (mRNA-LNP FGΔTM002) in which positions 162-172 and 187-199 of the F protein were replaced with a linker-linked sequence, mRNA-LNP FGΔTM001 was fused with the transmembrane domain from the RSV F protein (mRNA-LNP FG-TM001), and mRNA-LNP FGΔTM002 was fused with the transmembrane domain from the RSV F protein (mRNA-LNP FG-TM002).

[0056] Figure 2 shows the anti-F antibody titers in the serum of C57BL / 6J mice immunized twice. Compared with protein RSV FG+Alum (a protein antigen containing the same amino acid sequence encoded by the mRNA of mRNA-LNP FGΔTM 001 and the Alum-based adjuvant Adju-Phos (InvitroGen)), which was developed based on a prior patent (Patent Document 7), mRNA-LNP FG-TM 001 and mRNA-LNP FG-TM 002, which contain the transmembrane domain, showed a significant increase in anti-F antibody titers. On the other hand, mRNA-LNP FGΔTM 001 and mRNA-LNP FGΔTM 002, which do not contain the transmembrane domain, showed titers comparable to those of protein RSV FG+Alum.

[0057] Example 2: Anti-F antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 3. Similar to the results for the C57BL / 6J mice described above, mRNA-LNP FG-TM 001 and mRNA-LNP FG-TM 002, which contain a transmembrane domain, showed a significant increase in anti-F antibody titer compared to protein RSV FG+Alum, which was developed based on the prior patent (Patent Document 7). On the other hand, mRNA-LNP FGΔTM 001 and mRNA-LNP FGΔTM 002, which do not contain a transmembrane domain, showed titers comparable to protein RSV FG+Alum.

[0058] Example 3: Anti-G antibody titers in serum from C57BL / 6J mice immunized twice are shown in Figure 4. Compared with protein RSV FG+Alum, which was developed based on a prior patent (Patent Document 7), mRNA-LNP FG-TM 002 showed a significant increase in anti-G antibody titers. On the other hand, mRNA-LNP FGΔTM 001, mRNA-LNP FGΔTM 002, and mRNA-LNP FG-TM 001 showed titers comparable to protein RSV FG+Alum.

[0059] Example 4: Anti-G antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 5. Compared to protein RSV FG+Alum, which was developed based on a prior patent (Patent Document 7), mRNA-LNP FG-TM 002 showed a significant increase in anti-G antibody titer. On the other hand, mRNA-LNP FGΔTM 001 and mRNA-LNP FG-TM 001 showed levels comparable to protein RSV FG+Alum. Although mRNA-LNP FGΔTM 002 showed a tendency for anti-G antibody titer to increase, there was no significant difference.

[0060] Example 5: The neutralizing antibody titers (IC50) of pooled sera (N=5) from C57BL / 6J mice immunized twice were used to evaluate their efficacy. Figure 6 shows the neutralizing antibody titers (IC50) of pooled sera (N=5). Compared with protein RSV FG+Alum, which was developed based on a prior patent (Patent Document 7), mRNA-LNP FG-TM 001 and mRNA-LNP FG-TM 002, which contain a transmembrane domain, showed increased neutralizing antibody titers. The neutralizing antibody titers of mRNA-LNP FGΔTM 001 and mRNA-LNP FGΔTM 002, which do not contain a transmembrane domain, tended to increase compared with protein RSV FG+Alum, but were not as high as those of mRNA-LNP FG-TM 001 and mRNA-LNP FG-TM 002, which contain a transmembrane domain.

[0061] Example 6: To evaluate the efficacy of serum obtained by immunizing BALB / c mice twice, the neutralizing antibody titers (IC50) of pooled sera (N=5) are shown in Figure 7. Compared with protein RSV FG+Alum, which was developed based on a prior patent (Patent Document 7), mRNA-LNP FG-TM 001 and mRNA-LNP FG-TM 002, which contain a transmembrane domain, showed increased neutralizing antibody titers. The neutralizing antibody titers of mRNA-LNP FGΔTM 001 and mRNA-LNP FGΔTM 002, which do not contain a transmembrane domain, were comparable to those of protein RSV FG+Alum.

[0062] Example 7: Immunogenicity evaluation by mouse age in weeks is shown in Figure 8. In aged mice, mRNA-LNP FG-TM 001 containing a transmembrane domain had a higher anti-F antibody titer than protein RSV FG+Alum, which was developed based on a prior patent (Patent Document 7). Generally, immunogenicity is believed to be weaker in aged mice than in adult mice, and a comparison of the modalities of mRNA-LNP and protein antigens showed that adult mice had higher anti-F antibody titers than aged mice. Meanwhile, mRNA-LNP FG-TM 001 in aged mice had a higher anti-F antibody titer than protein RSV FG+Alum in adult mice.

[0063] Example 8: mRNA-LNP FG-TM-p27ΔFP 002, in which the G sequence insertion position of mRNA-LNP FG-TM 002 was altered to immediately before p27; mRNA-LNP FG-TM-C 002, in which the G sequence insertion position of mRNA-LNP FG-TM 002 was altered to the C-terminus; mRNA-LNP FG-TM 003, in which the G sequence of mRNA-LNP FG-TM 002 was altered to a sequence in which positions 162-172, 187-199, and 162-172 were linked in that order with a linker (EAAAKDFHFEVFNFVPEAAAKKRIPNKKPGKKTTEAAAKDFHFEVFNFVPEAAAK: SEQ ID NO: 10); and mRNA-LNP FG-TM-p27ΔFP, in which the G sequence insertion position of mRNA-LNP FG-TM 003 was altered to immediately before p27. We created 003 and investigated the effect of the G sequence insertion position and G sequence ligation. ΔFP indicates deletion of 137-146 of the FP sequence.

[0064] For each mutant created in Example 8, the anti-F antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 9 (GMT, error bar: 95% CI, n = 5-8). Compared to protein RSV FG + Alum, mRNA-LNP FG-TM 002 (2.5, 5, 10 μg) showed a significant increase in anti-F antibody titers, confirming reproducibility. Furthermore, when compared with at least one dose of mRNA-LNP FG-TM 002, mRNA-LNP FG-TM-p27ΔFP 002, mRNA-LNP FG-TM-C 002, mRNA-LNP FG-TM 003, and mRNA-LNP FG-TM-p27ΔFP 003 showed increased anti-F antibody titers. Therefore, it can be said that changing the G sequence insertion position immediately before p27 or at the C-terminus and changing from two G sequences to three G sequences improved the anti-F antibody induction ability.

[0065] Example 9: For each mutant created in Example 8, the anti-G antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 10 (GMT, error bar: 95% CI, n = 5-8). Compared to mRNA-LNP FG-TM 002, the anti-G antibody titers of mRNA-LNP FG-TM-p27ΔFP 002, mRNA-LNP FG-TM 003, and mRNA-LNP FG-TM-p27ΔFP 003 were similar. Under the 2.5 μg / dose condition, the anti-G antibody titers of mRNA-LNP FG-TM-p27ΔFP 002, mRNA-LNP FG-TM 003, and mRNA-LNP FG-TM-p27ΔFP 003 tended to be higher than those of mRNA-LNP FG-TM 002. Furthermore, the anti-G antibody titer of mRNA-LNP FG-TM-C 002 was similar to that of protein FG+Alum, which is thought to be due to the inserted G sequence being localized around the cell membrane or in the cytoplasm.

[0066] [Example 10] For each mutant created in Example 8, the neutralizing antibody titers of pooled sera obtained by immunizing BALB / c mice twice are shown in Figure 11 (GMT). Compared to protein RSV FG+Alum, mRNA-LNP FG-TM 002 tended to have a higher neutralizing antibody titer, confirming reproducibility. Compared to mRNA-LNP preF, mRNA-LNP FG-TM-p27ΔFP 002, mRNA-LNP FG-TM-C 002, mRNA-LNP FG-TM 003, and mRNA-LNP FG-TM-p27ΔFP 003 tended to have higher neutralizing antibody titers.

[0067] Example 11: The effect of the type of transmembrane domain (TMD) was investigated. Specifically, mRNA FG-TM (H1N1), mRNA FG-TM (H2N2), and mRNA FG-TM (H3N2) were produced using the TMDs of the HA proteins of influenza viruses H1N1 (ILAIYSTVASSLVLLVSLGAI: SEQ ID NO: 11), H2N2 (ILAIYATVAGSLSLAIMMAGI: SEQ ID NO: 12), and H3N2 (ILWISFAISCFLLCVVLLGFI: SEQ ID NO: 13), which have hydrophobic amino acid ratios of 71.4%, 81.0%, and 90.5%, respectively. The G sequence in these mRNAs was G162-171, substituting for FP137-146. The possible effect of the type of linker connecting the TMD (H1N1, H2N2, and H3N2) to the F protein was also investigated. The linkers used were the GS linker (SEQ ID NO: 3) and the EAK linker (SEQ ID NO: 4). Furthermore, we investigated the possibility that the presence or absence of positions 551-574 of the F sequence, which is thought to be localized in the cytoplasm, may have an effect.

[0068] For each mutant produced in Example 11, the protein expression evaluation using flow cytometry is shown in Figure 12. The vertical axis represents FSC-H (cell size, main scale starts from 0 and is in 0.5 M increments), and the horizontal axis represents APC-A (reactivity with anti-F antibody, main scale starts from 10). １ , 10 ２ , 10 ３ , 10 ４ , 10 ５ , 10 ６ , 10 ７(Logarithmic scale). Without a linker, the reactivity with anti-F antibodies was highest in mRNA FG-TM (H3N2), followed by mRNA FG-TM (H2N2), and finally mRNA FG-TM (H1N1). It appears that the higher the hydrophobicity of the TMD, the higher the FG protein expression on the cell membrane. On the other hand, even for the less hydrophobic TMDs (H1N1 and H2N2) that showed low protein expression without a linker, protein expression increased when a GS linker was used. This is likely due to the increased hydrophobicity around the TMD caused by the GS linker adjacent to the TMD, anchoring the FG protein to the cell membrane. With the EAK linker, protein expression was improved compared to without a linker, but was lower than with the GS linker. Furthermore, protein expression was observed even when positions 551-574 of the F sequence were linked. Regardless of the presence or absence of positions 551-574 of the F sequence, the hydrophobicity of the TMD tended to affect protein expression.

[0069] [Example 12] To investigate the effect of different TMD types, mRNA FG-TM (human-derived) was produced using TMD derived from a human protein. Specifically, mRNA FG-TM (T1-1: Growth hormone receptor_P10912 (FPWLLIIIFGIFGLTVMLFVFLFS: SEQ ID NO: 14), T1-2: Low-density lipoprotein receptor_P01130 (ALSIVLPIVLLVFLCLGVFLLW: SEQ ID NO: 15), T1-3: Glycophorin-B_P06028 (PVVIILIILCVMAGIIGTILLI: SEQ ID NO: 16)) was used as Type I mRNA FG-TM (T2-1: Beta-1,3-galactosyltransferase 4_O96024 (LLLAALLLVIV: SEQ ID NO: 17), T2-2: Beta-1,3-galactosyltransferase 1_Q9Y5Z6 (CLYVLTVVCWASALWYLSIT: SEQ ID NO: 18)) was used as Type II mRNA FG-TM (T3-1: Linker for activation of The following genes were produced: T-cell family member 1 O43561 (ILVPCVLGLLLLPILAMLMALCV: SEQ ID NO: 19), T3-2: Lymphocyte transmembrane adapter 1 Q8IWV1 (IFSGFAGLLAILLVVAVFCIL: SEQ ID NO: 20), T3-3: Linker for activation of T-cell family member 2 Q9GZY6 (ELLWPGAALLVLLGVAASLCV: SEQ ID NO: 21), and T3-4: Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 Q9NWQ8 (LWGSLAAVAIFFVITFLIFLC: SEQ ID NO: 22). In all cases, the G sequence is at positions G162-171, replacing positions FP137-146.

[0070] For each mutant produced in Example 12, the protein expression evaluation using flow cytometry is shown in Figure 13. The vertical axis represents FSC-H (cell size, main scale starts from 0 and is in 0.5M increments), and the horizontal axis represents APC-A (reactivity with anti-F antibody, main scale starts from 10). １ , 10 ２ , 10 ３ , 10 ４ , 10 ５ , 10 ６ , 10 ７ (Logarithmic scale of the linker). Without a linker, FG protein expression was observed in all mRNA FG-TM (human origin) except T2-2. This is thought to be due to the fact that the hydrophobicity of T2-2 is 70%, while the hydrophobicity of the others is over 90%. When a GS linker was added, FG protein expression was observed in all mRNA FG-TM (human origin) except T2-1. Furthermore, when a GS linker was added to T2-2, which did not express FG protein without a linker, FG protein expression was observed. Interestingly, T2-1 has a TMD consisting of 11 amino acids with 100% hydrophobicity, and it was found that the addition of a GS linker reduced protein expression on the cell membrane.

[0071] Example 13 For each of the mutants created in Examples 11 and 12, the anti-F antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 14 (GMT, error bars: 95% CI, n = 5-8). Compared to Protein FG + Alum, TM(H1N1), TM(H2N2), and TM(T2-2) had lower anti-F antibody titers. On the other hand, TM-GS(H1N1), TM-GS(H2N2), and TM-GS(T2-2), which had GS linkers added to these sequences, showed higher anti-F antibody titers than Protein FG + Alum in at least one dose comparison. Furthermore, TM(H3N2), TM(T1-1), TM(T2-1), TM(T3-1), and TM(T3-4) had higher antibody titers than Protein FG + Alum in at least two dose comparisons. Furthermore, compared with mRNA-LNP preF, TM-GS (H1N1), TM-GS (H2N2), TM-GS (T2-2), TM (H3N2), TM (T1-1), TM (T2-1), TM (T3-1) and TM (T3-4) showed equivalent or higher anti-F antibody titers in at least one dose comparison.

[0072] [Example 14] The neutralizing antibody titers of pooled sera obtained by immunizing BALB / c mice twice with each of the mutants created in Examples 11 and 12 are shown in Figure 15 (GMT). TM(H1N1), TM(H2N2), and TM(T2-2) showed low titers. On the other hand, TM-GS(H1N1), TM-GS(H2N2), and TM-GS(T2-2), which were derived by affixing a GS linker to these sequences, showed similar neutralizing antibody titers to TM-GS(H1N1), TM-GS(H2N2), TM-GS(T2-2), TM(H3N2), TM(T1-1), TM(T2-1), TM(T3-1), and TM(T3-4) at at least one dose comparison. Relatively high neutralizing antibody titers were detected in all groups with a TMD hydrophobicity ratio of 90% or higher, or in groups with a TMD hydrophobicity ratio of less than 90% but affixed with a GS linker.

[0073] Example 15: The effects of converting RSV F protein to the preF form were investigated. Specifically, an FG protein (preFG) was created based on an F protein containing DS-Cav1 mutations (S155C, S290C, S190F, V207L) that maintain a stable preF structure. Mutants of preFG were created, each with a different TMD, the type and insertion position of the G sequence, and a partial deletion of the FP region. BALB / c mice were immunized twice, and pooled sera were analyzed.

[0074] Figure 16 (GMT, error bars: 95% CI, n = 5-8) shows the anti-F antibody titers in the serum of BALB / c mice immunized twice with each mutant prepared in Example 15. Compared to Protein FG + Alum, TMD-free preFG-C 001, preFG-C 002, preFG-C 001ΔFP, preFG-C 002ΔFP, preFG 001, and preFG 002 showed equivalent or higher anti-F antibody titers at high doses (5 μg / dose, 10 μg / dose). On the other hand, preFG-TM-C 001, preFG-TM-C 002, preFG-TM-C 001ΔFP, preFG-TM-C 002ΔFP, preFG-TM 001, and preFG-TM 002, which contain the TMD, showed higher anti-F antibody titers than Protein FG + Alum even at a low dose (2.5 μg / dose), and were equal to or higher than mRNA-LNP preF. In particular, preFG-TM 002 showed higher anti-F antibody titers than mRNA-LNP preF. Furthermore, mRNA-LNP preF contains the TMD region of the F protein.

[0075] Example 16 For each mutant created in Example 15, the anti-G antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 17 (GMT, error bars: 95% CI, n = 5-8). Compared to Protein FG + Alum, the TMD-free mutants preFG-C 001, preFG-C 002, preFG-C 001ΔFP, preFG-C 002ΔFP, and preFG 002 had anti-G antibody titers equal to or higher than Protein FG + Alum. In particular, preFG-C 001ΔFP, preFG-C 002ΔFP, and preFG 002 had higher anti-G antibody titers than Protein FG + Alum in at least one dose comparison. Among the TMD-containing mutants, preFG-TM 002 had higher anti-G antibody titers than Protein FG + Alum in all dose comparisons. On the other hand, the mutants preFG-TM-C 001, preFG-TM-C 002, preFG-TM-C 001ΔFP, and preFG-TM-C 002ΔFP, which contain both the TMD and a G sequence at the C-terminus, showed lower anti-G antibody titers than Protein FG + Alum. This is likely due to the G sequence added after the TMD, which may have led to the expression of the G protein at the cell membrane or in the cytoplasm. Indeed, preFG-TM 002, which contains both the TMD and two linked G sequences inserted at positions other than the C-terminus, showed higher anti-G antibody titers than Protein FG + Alum.

[0076] [Example 17] For each mutant created in Example 15, the neutralizing antibody titers of pooled sera obtained by immunizing BALB / c mice twice are shown in Figure 18 (GMT). The neutralizing antibody titers were as follows: preFG-TM-C 001 > preFG-C 001, preFG-TM-C 002 > preFG-C 002, preFG-TM-C 001ΔFP > preFG-C 001ΔFP, preFG-TM-C 002ΔFP > preFG-C 002ΔFP, preFG-TM 001 > preFG 001, and preFG-TM 002 > preFG 002. Therefore, the neutralizing antibody titers of all preFG mutants were improved by the addition of TMD.

[0077] [Example 18] Based on Examples 1 to 17, mutants with relatively high neutralizing antibody titers against the RSV A strain were selected, and the neutralizing antibody titers against the RSV B strain were measured and shown in Figure 19 (GMT, n = 5-8). Compared to Protein FG + Alum, preFG-TM 002, FG-TM 003, FG-TM-p27ΔFP 003, TM_H3N2, TM_GS_H1N1, and TM_T3-4 exhibited high neutralizing antibody titers. In particular, FG-TM 003, FG-TM-p27ΔFP 003, TM_H3N2, TM_GS_H1N1, and TM_T3-4 exhibited neutralizing antibody titers higher than those of mRNA-LNP preF.

[0078] Example 19: In vivo protection against infection was examined. BALB / c mice were challenged with RSV A strain after two immunizations, and the results of evaluating protection against infection are shown in Figure 20 (GMT, error bar: 95% CI, n = 8). FG-TM-p27ΔFP 003 demonstrated superior protection against infection compared to protein FG + Alum, preF (ALC-0315), and preF (SM-102) in terms of GMT values. The percentage of mice with virus copy numbers below the limit of detection (LOD) in lung tissue is also shown in Figure 21. FG-TM-p27ΔFP 003 demonstrated a superior protective effect against infection compared to protein FG + Alum, preF (ALC-0315), and preF (SM-102).

[0079] Example 20 Based on the results of Examples 1 to 17, we created several mutants in which SEQ ID NO: 10, consisting of three G sequences linked by a linker, was inserted immediately before p27. We also investigated the effects of the TMD type, deletion of FP sequence 137-146, and preF-type FG in these mutants. The anti-F antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 22 (GMT, error bar: 95% CI, n = 6-8). Compared with protein FG + Alum and FGΔTM-p27ΔFP 003 (secreted form) which does not contain a TMD, the mutants FG-TM-p27ΔFP 003, TM-H3N2-p27ΔFP 003, TM-GS-H1N1-p27ΔFP 003, TM-3-4-p27ΔFP 003, and preFG-TM-p27ΔFP, which contain a TMD and have ΔFP 137-146, all had higher anti-F antibody titers at 5 μg / dose. On the other hand, the mutants FG-TM-p27 003, TM-H3N2-p27 003, TM-3-4-p27 003, and preFG-TM-p27, which contain the FP sequence 137-146, at 5 μg / dose were superior to protein FG + Alum but were similar to FGΔTM-p27ΔFP 003 (secreted type).

[0080] Example 21 For each of the mutants produced in Example 20, the anti-G antibody titers in the serum of BALB / c mice immunized twice are shown in Figure 23 (GMT, error bar: 95% CI, n = 6-8). Compared to protein FG + Alum, the mutants FG-TM-p27ΔFP 003, TM-H3N2-p27ΔFP 003, TM-GS-H1N1-p27ΔFP 003, TM-3-4-p27ΔFP 003, and preFG-TM-p27ΔFP, which contain a TMD and ΔFP 137-146, all had higher anti-G antibody titers at 5 μg / dose. Furthermore, the mutants FG-TM-p27 003, TM-H3N2-p27 003, TM-3-4-p27 003, and preFG-TM-p27, which contain the FP sequence 137-146, tended to have lower activity than mutants lacking the FP sequence positions 137-146.

[0081] Example 22 The neutralizing antibody titers of pooled sera obtained by immunizing BALB / c mice twice with each of the mutants prepared in Example 20 are shown in Figure 24 (GMT, error bars: 95% CI, n = 6-8). Compared with protein FG + Alum and TMD-free FGΔTM-p27ΔFP 003 (secretor), the mutants FG-TM-p27ΔFP 003, TM-H3N2-p27ΔFP 003, TM-GS-H1N1-p27ΔFP 003, TM-3-4-p27ΔFP 003, and preFG-TM-p27ΔFP, which contain TMD and ΔFP 137-146, all showed higher neutralizing antibody titers at 5 μg / dose. Furthermore, among the mutants containing FP sequence 137-146, FG-TM-p27003 and TM-GS-H1N1-p27003 also exhibited higher 5 μg / dose activity than protein FG + Alum and FGΔTM-p27ΔFP003 (secreted form), which do not contain TMD. TM-3-4-p27003 and preFG-TM-p27003 also tended to exhibit higher 5 μg / dose activity than FGΔTM-p27ΔFP003 (secreted form).

[0082] The present invention is useful in the field of medicine, particularly in the field of vaccines.

Claims

1. An mRNA encoding a chimeric protein (RSV FG chimeric protein) of the respiratory syncytial virus (RSV), which uses a sequence in which the RSV F protein ectodomain is fused with the transmembrane domain as a basic backbone, with part of the basic backbone replaced with part of the CCD sequence of the RSV G protein, or an mRNA encoding an RSV FG chimeric protein in which part of the CCD sequence is added to the basic backbone.

2. The mRNA of claim 1, wherein a portion of the CCD sequence encodes a sequence in which multiple sequences of the RSV G protein sequence of SEQ ID NO: 1, excluding the CX3C motif, are linked together.

3. The mRNA according to claim 1, wherein a portion of the CCD sequence encodes a sequence in which two or three sequences from positions 162 to 171 and 187 to 197 of the RSV G protein of SEQ ID NO: 1 are linked together.

4. The mRNA according to claim 1, wherein a portion of the CCD sequence encodes a sequence in which two or three sequences from positions 162-172 and 187-199 of the RSV G protein of SEQ ID NO: 1 are linked together.

5. The mRNA of claim 1, wherein a portion of the CCD sequence encodes a sequence obtained by linking the RSV G protein sequence of SEQ ID NO: 1, excluding the CX3C motif, with a linker.

6. The mRNA according to claim 5, wherein the linker connecting multiple sequences excluding the CX3C motif is GGGGS (SEQ ID NO: 3) or EAAAK (SEQ ID NO: 4).

7. The mRNA of claim 1, wherein a portion of the FP domain of the RSV F protein is replaced with a portion of a CCD sequence encoding a sequence in which multiple sequences, excluding the CX3C motif, of the RSV G protein sequence of SEQ ID NO: 1 are linked together.

8. The sequence corresponding to positions 137-146 of the RSV F protein set forth in SEQ ID NO: 2 is replaced with a portion of a CCD sequence encoding a sequence in which multiple sequences, excluding the CX3C motif, of the RSV G protein sequence of SEQ ID NO: 1 are linked, or the sequence corresponding to positions 137-146 of the RSV F protein is deleted, and a portion of the CCD sequence encoding a sequence in which multiple sequences, excluding the CX3C motif, of the RSV G protein sequence of SEQ ID NO: 1 are linked is added, according to claim 1, the mRNA.

9. The mRNA of claim 1, wherein a portion of a CCD sequence encoding a sequence in which multiple sequences excluding the CX3C motif of the RSV G protein sequence of SEQ ID NO: 1 are linked to the N-terminus or C-terminus of the transmembrane region fused to the RSV F protein ectodomain is added.

10. The mRNA of claim 1, wherein a portion of a CCD sequence encoding a sequence in which multiple sequences excluding the CX3C motif of the RSV G protein sequence of SEQ ID NO: 1 are linked between the p27 and FP domains of the RSV F protein.

11. The mRNA of claim 1, comprising a portion of the CCD sequence of claim 2 added to the C-terminus of the F2 domain of the RSV F protein.

12. The mRNA of claim 1, wherein a portion of a CCD sequence encoding a sequence in which multiple sequences excluding the CX3C motif of the RSV G protein sequence of SEQ ID NO: 1 are linked to the RSV F protein ectodomain or transmembrane region via a linker.

13. The mRNA according to claim 11, wherein the linker connecting the basic skeleton and a portion of the CCD sequence is GGGGS (SEQ ID NO: 3) or EAAAK (SEQ ID NO: 4).

14. The mRNA of claim 1, wherein the ectodomain of the RSV F protein encodes an amino acid sequence having 90% or more sequence identity with the amino acid sequence of positions 1-513 of SEQ ID NO:

2.

15. The mRNA of claim 13, wherein the ectodomain of the RSV F protein encodes an amino acid sequence containing a pre-F mutation.

16. The mRNA of claim 1, wherein the transmembrane domain encodes an amino acid sequence derived from a viral protein.

17. The mRNA of claim 1, wherein the transmembrane region encodes an amino acid sequence derived from the RSV F protein.

18. The mRNA of claim 1, wherein the transmembrane region encodes an amino acid sequence derived from an influenza virus HA protein.

19. The mRNA of claim 18, wherein the influenza virus HA protein is an H1N1, H2N2, or H3N2 HA protein.

20. The mRNA of claim 1, wherein the transmembrane region encodes an amino acid sequence derived from a human protein.

21. The mRNA of claim 20, wherein the human protein is selected from the group consisting of growth hormone receptor, low-density lipoprotein receptor, glycophorin-B, beta-1,3-galactosyltransferase 1, beta-1,3-galactosyltransferase 4, linker for activation of T-cell family member 1, lymphocyte transmembrane adapter 1, linker for activation of T-cell family member 2, and phosphoprotein associated with glycosphingolipid-enriched microdomains 1.

22. The mRNA according to claim 1, wherein the transmembrane region has a hydrophobic amino acid ratio of 70% or more.

23. The mRNA of claim 1, wherein the transmembrane region has a hydrophobic amino acid ratio of 80% or more.

24. The mRNA of claim 1, wherein the transmembrane region has a hydrophobic amino acid ratio of 90% or more.

25. The mRNA of claim 15, wherein the F protein has a mutation corresponding to S155C, S290C, S190F, or V207L ​​in SEQ ID NO: 2, or a mutation corresponding to T103C, I148C, S190I, and D486S in SEQ ID NO:

2.

26. DNA for synthesizing the mRNA according to any one of claims 1 to 25.

27. A protein comprising an amino acid sequence according to any one of claims 1 to 25.

28. A RSV vaccine comprising the mRNA of any one of claims 1 to 25.

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