Respiratory syncytial virus RNA vaccine

Abstract

The present disclosure provides a respiratory syncytial virus (RSV) vaccine, comprising a messenger RNA (mRNA) containing an open reading frame (ORF) encoding a RSV F protein antigen, and a method for inducing an immune response by administering the vaccine.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 276,233, filed November 5, 2021, and European Patent Application No. 22315065.7, filed March 16, 2022, each of which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and the leading cause of respiratory disease in the elderly. RSV remains a vaccine need that remains unmet despite decades of research. Recent clinical programs using RSV F antigen in its postfusion conformation have failed to induce sufficient efficacy in adults. See Faloon et al. (2017) JID 216:1362-1370. However, RSV F antigen stabilized in the prefusion conformation may induce a better neutralizing response than the postfusion antigen that failed in the clinic. Summary of the Invention [Problem to be solved by the invention]

[0003] RNA-based vaccines (e.g., mRNA vaccines) have recently emerged as an effective vaccine type against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) mRNA vaccines have demonstrated a rapid, safe, and cost-effective production process. Often combined with a delivery vehicle such as lipid nanoparticles (LNPs), COVID-19 mRNA vaccines can achieve high efficacy. Due to the lack of available effective RSV vaccines, there is a need for an RNA-based RSV vaccine that induces a strong immune response against the RSV pre-fusion F protein for potent neutralization of RSV infection. [Means for solving the problem]

[0004] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity (e.g., 98%, 99% or 100% identity) to SEQ ID NO:3, or consists of the amino acid sequence of SEQ ID NO:3.

[0005] In a specific embodiment, the RSV F protein antigen is a pre-fusion protein.

[0006] In certain embodiments, the ORF is codon optimized.

[0007] In certain embodiments, the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR) and at least one polyadenylation (poly(A)) sequence.

[0008] In certain embodiments, the mRNA comprises at least one chemical modification.

[0009] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0010] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the uracil nucleotides in the ORF are chemically modified.

[0011] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0012] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0013] In certain embodiments, the chemical modification is N1-methylpseudouridine.

[0014] In certain embodiments, the mRNA is formulated in a lipid nanoparticle (LNP).

[0015] In certain embodiments, the LNP comprises at least one cationic lipid.

[0016] In certain embodiments, the cationic lipids are biodegradable. In certain embodiments, the cationic lipids are not biodegradable.

[0017] In certain embodiments, the cationic lipid is cleavable. In certain embodiments, the cationic lipid is not cleavable.

[0018] In certain embodiments, the cationic lipid is selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10 and GL-HEPES-E3-E12-DS-3-E14.

[0019] In a particular embodiment, the cationic lipid is cKK-E10.

[0020] In a particular embodiment, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

[0021] In certain embodiments, the LNPs further comprise polyethylene glycol (PEG)-conjugated (PEGylated) lipids, cholesterol-based lipids, and helper lipids.

[0022] In certain embodiments, the LNPs comprise cationic lipids in a molar ratio of 35% to 55%; polyethylene glycol (PEG)-conjugated (PEGylated) lipids in a molar ratio of 0.25% to 2.75%; cholesterol-based lipids in a molar ratio of 20% to 45%; and helper lipids in a molar ratio of 5% to 35%, all molar ratios relative to the total lipid content of the LNP.

[0023] In certain embodiments, the LNP comprises a 40% molar ratio of cationic lipid, a 1.5% molar ratio of PEGylated lipid, a 28.5% molar ratio of cholesterol-based lipid, and a 30% molar ratio of helper lipid.

[0024] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0025] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0026] In certain embodiments, the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0027] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 40%, DMG-PEG2000 in a molar ratio of 1.5%, cholesterol in a molar ratio of 28.5%, and DOPE in a molar ratio of 30%.

[0028] In certain embodiments, the LNPs comprise cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%.

[0029] In certain embodiments, the LNPs have an average diameter between 30 nm and 200 nm, In certain embodiments, the LNPs have an average diameter between 80 nm and 150 nm.

[0030] In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 80% identity to the nucleic acid sequence set forth in SEQ ID NO:6.

[0031] In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 80% identity to the nucleic acid sequence set forth in SEQ ID NO:14.

[0032] In certain embodiments, the mRNA comprises the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO:6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO:11; and (v) poly(A) tail Includes.

[0033] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine, comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the mRNA comprises the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO:6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO:11; and (v) poly(A) tail the mRNA is formulated into lipid nanoparticles (LNPs) containing 40% molar ratio of GL-HEPES-E3-E12-DS-4-E10, 1.5% molar ratio of DMG-PEG2000, 28.5% molar ratio of cholesterol, and 30% molar ratio of DOPE.

[0034] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine, comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the mRNA comprises the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO:6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO:11; and (v) poly(A) tail the mRNA contains cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5% and DOPE at a molar ratio of 30%.

[0035] In another aspect, the disclosure provides a method of inducing an immune response against RSV or protecting a subject against RSV infection, comprising administering to a subject an RSV vaccine as described above.

[0036] In a specific embodiment, the subject has a higher serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:1.

[0037] In certain embodiments, a subject has a comparable serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a protein RSV vaccine.

[0038] In certain embodiments, the protein RSV vaccine is co-administered with an adjuvant.

[0039] In a specific embodiment, the RSV vaccine increases the serum concentration of antibodies having binding specificity for site Φ of the RSV F protein.

[0040] In certain embodiments, the subject has a lower serum concentration of antibodies having binding specificity for site I or site II of the RSV F protein after administration of the RSV vaccine compared to a subject administered a RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:2.

[0041] In certain embodiments, the RSV vaccine increases serum concentrations of neutralizing antibodies in subjects with pre-existing RSV immunity.

[0042] In another aspect, the disclosure provides a RSV vaccine for use in eliciting an immune response to RSV or protecting a subject against RSV infection, comprising administering to a subject the RSV vaccine described above.

[0043] In certain embodiments, the RSV vaccines described above are used in the manufacture of a medicament for eliciting an immune response against RSV or for protecting a subject against RSV infection.

[0044] The above and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0045] [Figure 1A-1B] Western blot images of FD1, FD2 and FD3 proteins from transfected cells are shown. HEK293FT cells seeded in 6-well plates were transfected with 3 μg of mRNA using the MIRUS kit and cell lysates (FD1 and FD3) or cell supernatants (FD2) were harvested 24 hours after transfection (Figure 1A). FD1 mRNA was assessed by cell-free means using an in vitro transcription (IVT) kit to produce protein and compared with protein from FD1 mRNA transfected cells. Harvested samples were subjected to Western blot analysis and membranes were stained with 5353C75 monoclonal antibody (Figure 1B). [Figure 2A-2B]Western blots of transfected HEK versus nucleofected HSkMC are shown. HEK293FT cells seeded in 6-well plates were transfected with 5 μg of mRNA using the MIRUS kit and cell lysates (FD1 and FD3) or cell supernatants (FD2) were harvested 24 hours after transfection (Figure 2A). HSkM cells were nucleofected with 5 μg of mRNA using the Amaxa basic nucleofector kit and cell lysates (FD1 and FD3) or cell supernatants (FD2) were harvested 24 hours after nucleofection (Figure 2B). Harvested samples were subjected to Western blot analysis and membranes were stained with 5353C75 monoclonal antibody. [Diagram 3] Immunostaining of transfected HEK cells. HEK293FT cells seeded in 24-well plates were transfected with 5 μg of mRNA using the MIRUS kit. 24 hours after transfection, monoclonal antibodies D25 and Synagis were added to the plates along with fluorescently tagged secondary antibodies and imaged using Celigo. [Figure 4A-4B] 4A-4B show the immunogenicity of selected RSV antigens in naive non-human primates (NHPs). RSV F protein antibody titers (FIG. 4A) and RSV microneutralization titers (FIG. 4B) were measured on days 0, 28, and 56 for each antigen composition. [Diagram 5] 1 shows the results of a competitive ELISA using sera from NHPs immunized with RSV antigens selected against three known RSV F protein antibodies, D25, Synagis (palivizumab), and 131-2a. [Figure 6A-6B]Figure 6 shows the effect of pre-immune boosting in cynomolgus monkeys by RSV F ELISA and RSV microneutralization assay. Anti-RSV-F antibody titers in boosted monkeys were measured by end-point ELISA using DS-Cav1 Pre-F protein as binding antigen and detected with goat anti-human IgG. Readings from individual animals (n=6) are shown for D0, D14 and D28 time points with GMT + / - 95% confidence intervals (value above each value = GMT). Statistical analysis was performed using two-way ANOVA with Tukey's post-hoc test for multiple comparisons (Figure 6A). RSV neutralizing antibody titers were measured by microneutralization assay using WT A2-GFP RSV strain mixed with serially diluted sera from vaccinated monkeys on 96-well plates of Vero cells. Titers were determined by calculating the reverse decrease in fluorescent foci after 24 hours of incubation. Readings from individual animals (n=6) are shown with GMT + / - 95% confidence intervals (value above each value = GMT) for D0, D14 and D28 time points. Statistical analysis was performed using two-way ANOVA with Tukey's post-hoc test for multiple comparisons (Figure 6B). [Figure 7] Figure 1 shows RSV F protein antibody titers in NHPs immunized with FD3 F protein expressing mRNA. The mRNA was delivered with lipid nanoparticles (LNPs) containing one of several cationic lipids. For each antigen composition, antibody titers were measured on days 0, 21, and 35. [Figure 8] Figure 1 shows RSV neutralization titers in NHPs immunized with FD3 F protein expressing mRNA. The mRNA was delivered with lipid nanoparticles (LNPs) containing one of several cationic lipids. For each antigen composition, antibody titers were measured on days 0, 21, and 35. [Figure 9A-9B] 9A-9B show the immunogenicity of selected RSV antigens in naive mice. RSV F protein antibody titers (FIG. 9A) and RSV microneutralization titers (FIG. 9B) were measured on days 0, 21, and 35 for each antigen composition. [Figure 10A-10B]Pre-F IgG titers (FIG. 10A) and pre-F / post-F binding ratios (FIG. 10B) are shown using selected RSV antigens in the Modular Immune In Vitro Construct (MIMIC®) system. [Figure 11] 1 shows anti-RSV neutralizing titers in the MIMIC® system from donors with pre-existing RSV immunity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present disclosure relates, inter alia, to novel RNA (e.g., mRNA) compositions encoding RSV F proteins and vaccination methods therewith. In particular, the present disclosure relates to mRNAs encoding RSV Pre-F proteins formulated in lipid nanoparticles (LNPs).

[0047] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, however, methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In the event of a conflict, the present specification, including definitions, will control. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and embodiments, the terms "having" and "including" or variations such as "having", "having", "including" or "including" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited in this specification, this citation is not an admission that any of these documents form part of the general knowledge in the art.

[0048] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0049] Furthermore, as used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0050] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0052] Units, prefixes and symbols are shown in the format accepted by the International System of Units (SI). Numeric ranges include the numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Thus, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0053] The term "approximately" or "about" is used herein to mean approximately, roughly, in the region, or within the region. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above and below the stated value by, for example, a variance of 10%, up or down (higher or lower). In some embodiments, the term indicates a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" refers to a ±3% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±2% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±1% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.01% deviation from the indicated numerical value.

[0054] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. An mRNA may contain one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). The non-coding region of an mRNA includes the 5' cap, 5' untranslated region (UTR), 3'UTR and poly(A) tail. mRNA can be purified from natural sources, produced using recombinant expression systems (e.g., in vitro transcription), and optionally purified or chemically synthesized.

[0055] As used herein, the term "antigenic site Φ" ​​or "site Φ epitope" refers to a site located at the apex of the pre-fusion RSV F trimer, including amino acid residues 62-69 and 196-209 of wild-type RSV F (SEQ ID NO: 1). The site Φ epitope is a binding site for antibodies with specificity for pre-fusion RSV F, such as D25 and AM14, whose binding to the site Φ epitope blocks cell surface attachment of RSV (see, e.g., McLellan et al., Science, 340(6136):1113-1117, 2013). Recombinant human anti-RSV antibody D25 (Creative Biolabs®; Catalog No. PABL-322) and recombinant human anti-RSV antibody AM14 (Creative Biolabs®; Catalog No. PABL-321), respectively, are commercially available.

[0056] As used herein, the term "antigen stability" refers to the stability of an antigen over time or in solution.

[0057] As used herein, the term "cavity-filling substitution" refers to an engineered hydrophobic substitution that fills the cavity present in the pre-fusion RSV F trimer.

[0058] As used herein, the term "F protein" or "RSV F protein" refers to a protein of RSV that plays a role in promoting fusion of the viral envelope with the host cell membrane during viral entry.

[0059] As used herein, the term "RSV F polypeptide" or "F polypeptide" refers to a polypeptide that contains at least one epitope of the F protein.

[0060] As used herein, the term "glycan addition" refers to the addition of a mutation that introduces a glycosylation site not present in wild-type RSV F, which can be engineered to increase construct expression, increase construct stability, or block epitopes shared between pre-fusion and post-fusion conformations. Modified proteins containing glycan addition will have more glycosylation and therefore a higher molecular weight. Glycan addition can reduce the extent to which the RSV F polypeptide induces antibodies against the post-fusion conformation of RSV F.

[0061] As used herein, the term "intrapromer stabilizing substitution" refers to an amino acid substitution in RSV F that stabilizes the pre-fusion conformation by stabilizing the interaction within the RSV F trimer promoter.

[0062] As used herein, the term "inter-promoter stabilizing substitution" refers to an amino acid substitution in RSV F that stabilizes the pre-fusion conformation by stabilizing the interaction of the RSV F trimer promoter.

[0063] As used herein, the term "protease cleavage" refers to the proteolysis (sometimes referred to as "clipping") of a sensitive residue (eg, lysine or arginine) in a polypeptide sequence.

[0064] As used herein, the term "post-fusion" with respect to RSV F refers to a stable conformation of RSV F that occurs after the merging of the viral and cellular membranes.

[0065] As used herein, the term "prefusion" with respect to RSV F refers to the conformation of RSV F adopted prior to virus-cell interaction.

[0066] As used herein, the term "protomer" refers to a structural unit of an oligomeric protein. In the case of RSV F, the individual units of the RSV F trimer are protomers.

[0067] As used herein, the term "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. Thus, an N-glycan is formed by the process of N-glycosylation. The glycan may be a polysaccharide.

[0068] As used herein, the term "glycosylation" refers to the addition of sugar units to a protein.

[0069] As used herein, the term "immune response" refers to the reaction of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage, or polymorphonuclear cell, to a stimulus, such as an antigen or a vaccine. The immune response can include any cell of the body that participates in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses.

[0070] As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (e.g., prevents an infectious disease or prevents the development of a disease associated with an infectious disease). Methods of measuring an immune response include, for example, measuring lymphocyte (such as B cells or T cells) proliferation and / or activity, cytokine or chemokine secretion, inflammation, antibody production, and the like.

[0071] As used herein, an "antibody response" is an immune response in which antibodies are produced.

[0072] As used herein, "antigen" refers to an agent that, when exposed to or administered to an organism, elicits an immune response and / or binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including production of antigen-specific antibodies). Alternatively or in addition, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism's species. In some embodiments, the antigen induces an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of members of the target species. In some embodiments, the antigen binds to an antibody and / or a T cell receptor and may or may not induce a specific physiological response in an organism. In some embodiments, for example, the antigen binds to an antibody and / or a T cell receptor in vitro, regardless of whether such interactions occur in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity. The antigen includes a RSV polypeptide encoded by an mRNA described herein.

[0073] As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed. Water-in-oil or oil-in-water emulsions in which the antigen solution is emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Killed mycobacteria are sometimes included to further enhance antigenicity (e.g., Freund's complete adjuvant). Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants can also include biological molecules such as Toll-like receptor (TLR) agonists and costimulatory molecules.

[0074] As used herein, an "antigenic RSV polypeptide" refers to a polypeptide that contains all or a portion of an RSV amino acid sequence of sufficient length that the molecule is antigenic to RSV.

[0075] As used herein, a "subject" refers to any member of the animal kingdom. In some embodiments, a "subject" refers to a human. In some embodiments, a "subject" refers to a non-human animal. In some embodiments, a subject includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, an insect, and / or a worm. In certain embodiments, a non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In some embodiments, a subject is an adult, an adolescent, or an infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject." In certain exemplary embodiments, the subject is a premature infant (e.g., less than 37 weeks gestation), a neonate (e.g., 0-27 days old), an infant or toddler (e.g., 28 days to 23 months old), a child (e.g., 2-11 years old), an adolescent (e.g., 12-17 years old), an adult (e.g., 18-50 years old or 18-64 years old), or an elderly person (e.g., 65 years old or older). In exemplary embodiments, the subject is 18-50 years old. In other exemplary embodiments, the subject is an elderly person (e.g., an adult 60 years old or older).

[0076] As used herein, the term "vaccination" or "vaccinate" refers to the administration of a composition intended to generate an immune response, for example against a disease-causing agent. Vaccination can occur before, during and / or after exposure to a disease-causing pathogen and / or before, during and / or after the onset of one or more symptoms, in some embodiments before, during and / or immediately after exposure to a disease-causing pathogen. In some embodiments, vaccination involves multiple administrations of the vaccinating composition at appropriate time intervals.

[0077] This disclosure describes nucleic acid sequences (eg, DNA and RNA sequences) and amino acid sequences that have a degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence).

[0078] "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0079] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is usually performed by comparing said sequences over a segment or "window of comparison" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs which employ said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0080] The percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying this result by 100.

[0081] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, in consecutive nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0082] A nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional characteristic of the given sequence, e.g., in some instances is functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0083] As used herein, the term "kit" refers to a packaged set of one or more compounds or compositions and one or more associated materials, such as solvents, solutions, buffers, instructions, or desiccants, or other associated components.

[0084] II. RNA The RSV vaccine of the present disclosure may comprise at least one ribonucleic acid (RNA) comprising an ORF encoding a RSV F protein antigen. In certain embodiments, the RNA is a messenger RNA (mRNA) comprising an ORF encoding a RSV F protein antigen. In certain embodiments, the RNA (e.g., mRNA) further comprises at least one 5'UTR, 3'UTR, poly(A) tail, and / or 5' cap.

[0085] II.A.5' Cap The 5' cap of an mRNA provides resistance to nucleases found in most eukaryotic cells and can promote translation efficiency. Several types of 5' caps are known: 7-methylguanosine cap ("m-cap"); 7 The second transcribed nucleotide (also called "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0086] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, generating a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0087] 5'-capping of polynucleotides can be completed simultaneously during in vitro transcription reactions using the following chemical RNA cap analogs to generate 5'-guanosine cap structures according to manufacturer's protocols: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-capping of modified RNAs can be completed post-transcriptionally using vaccinia virus capping enzyme to generate Cap 0 structures. m7G(5')ppp(5')G. Cap 1 structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structures can be generated from Cap 1 structures, followed by 2'-O-methylation of the 5'-penultimate nucleotide using 2'-O methyl-transferase. Cap 3 structures can be generated from Cap 2 structures, followed by 2'-O-methylation of the 5'-most terminal nucleotide using 2'-O methyl-transferase.

[0088] In certain embodiments, an mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU and m7G(5')ppp(5')(2'OMeG)pG.

[0089] In certain embodiments, the mRNA of the present disclosure comprises: [ka] Contains the 5' cap.

[0090] II.B. Untranslated Regions (UTRs) In some embodiments, an mRNA of the present disclosure comprises a 5' and / or 3' untranslated region (UTR). In an mRNA, the 5' UTR begins at the transcription initiation site and continues up to but not including the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0091] In some embodiments, the mRNAs disclosed herein may comprise a 5'UTR that includes one or more elements that affect mRNA stability or translation. In some embodiments, the 5'UTR may be about 10-5,000 nucleotides in length. In some embodiments, the 5'UTR may be about 50-500 nucleotides in length. In some aspects, the 5'UTR may be at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 60 ... The length is about 50 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides.

[0092] In some embodiments, the mRNAs disclosed herein may include a 3'UTR that includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the location of the mRNA in a cell, or one or more binding sites for an miRNA. In some embodiments, the 3'UTR may be 50-5,000 nucleotides in length or longer. In some embodiments, the 3'UTR may be 50-1,000 nucleotides in length or longer. In some embodiments, the 3'UTR is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0093] In some embodiments, an mRNA disclosed herein may contain a 5' or 3' UTR that is derived from a gene that is different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0094] In certain embodiments, the 5' and / or 3' UTR sequences may be derived from stable (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) mRNAs to enhance the stability of the mRNA. For example, the 5' UTR sequence may include a subsequence or fragment of the CMV immediate early 1 (IE1) gene to improve nuclease resistance and / or improve half-life of the mRNA. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3' end or untranslated region of the mRNA. In general, these modifications include modifications made to improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA compared to the unmodified counterpart, e.g., to improve such mRNA resistance to in vivo nuclease digestion.

[0095] Exemplary 5'UTRs include sequences derived from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO: 18) (U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference).

[0096] In various embodiments, the 5'UTR may be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5' terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0097] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, supra).

[0098] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, supra).

[0099] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra).

[0100] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0101] In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 11. 5' UTRs and 3' UTRs are described in further detail in WO 2012 / 075040, which is incorporated herein by reference.

[0102] II.C. Polyadenylation Tail As used herein, the terms "poly(A) sequence," "poly(A) tail," and "poly(A) region" refer to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have a length of essentially 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide that is different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides that is different from an adenosine nucleotide). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 19).

[0103] As used herein, a "poly(A) tail" typically relates to RNA. However, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0104] The poly(A) tail can comprise from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0105] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during in vitro transcription of the RNA. In certain embodiments, the poly(A) tail is obtained in vitro by common chemical synthesis methods without being transcribed from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using a commercially available polyadenylation kit and corresponding protocol or alternatively by using immobilized poly(A) polymerase, for example using the methods and means described in WO 2016 / 174271.

[0106] The nucleic acid may include a poly(A) tail obtained by enzymatic polyadenylation, with the majority of the nucleic acid molecules including from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0107] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA, and may further comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO 2016 / 091391.

[0108] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0109] In various embodiments, the nucleic acid can include at least one poly(C) sequence.

[0110] As used herein, the term "poly(C) sequence" is intended to mean a sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0111] II.D. Chemical modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may include at least one chemical modification. In some embodiments, the mRNA disclosed herein may include one or more modifications that typically enhance RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNAs contain modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxymethylaminomethyl) ... uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0112] In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0113] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0114] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0115] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0116] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the uracil nucleotides in the ORF are chemically modified.

[0117] The preparation of such analogs is described, for example, in U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642.

[0118] II.E. mRNA synthesis The mRNA disclosed herein may be synthesized according to any of a variety of methods. For example, the mRNA according to the present disclosure may be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101-14. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase and / or RNase inhibitor. The exact conditions may vary depending on the particular application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogenous mRNA suitable for therapeutic use. In some embodiments, mRNA provided from an in vitro transcription reaction may be desired, although other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants and / or animals.

[0119] In certain embodiments, the mRNA comprises the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO:6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO:11; and (v) poly(A) tail Includes.

[0120] In certain embodiments, the poly(A) tail has a length of about 10 to about 500 adenosine nucleotides.

[0121] III. RSV F Protein Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus belonging to the Pneumoviridae family. RSV can cause infection of the respiratory tract. RSV is an enveloped virus with glycoprotein (G protein), low molecular weight hydrophobic protein (SH protein) and fusion protein (F protein) on its surface.

[0122] The RSV F protein is involved in the fusion of the viral and host cell membranes and adopts at least three conformations (pre-fusion, intermediate and post-fusion conformations). In the pre-fusion conformation (pre-fusion, Pre-F), the F protein exists in a trimeric form with the major antigenic site φ exposed. Site φ serves as the primary target for neutralizing antibodies produced by RSV-infected subjects (see Coultas et al., Thorax. 74:986-993. 2019; McLellan et al., Science. 340(6136):1113-7. 2013). After binding to its target on the host cell surface, Pre-F undergoes a conformational change during which site φ is no longer exposed. Pre-F transitions to a transient intermediate conformation, allowing the F protein to be inserted into the host cell membrane, resulting in fusion of the viral and host cell membranes. A final conformational shift results in a more stable, extended form of the protein (post-fusion, post-F). Sites II and IV of the F protein are specific to post-F, while site I is present in both pre-F and post-F conformations (McLellan et al., J. Virol. 85(15):7788-7796. 2011).

[0123] Provided herein are RNAs (e.g., mRNAs) encoding antigenic RSV F polypeptides.

[0124] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3, or consists of the amino acid sequence of SEQ ID NO:3.

[0125] In some embodiments, the ORF is codon-optimized. As used herein, "codon-optimized" or "codon optimization" refers to the introduction of certain codons (in place of the respective wild-type codons that code for the same amino acid), which may be more favorable with respect to RNA stability and / or with respect to codon usage in a subject.

[0126] In some embodiments, an epitope of the RSV F protein shared between Pre-F and Post-F is blocked. Blocking the epitope reduces or eliminates the production of antibodies against the epitope when an RNA (e.g., mRNA) encoding an antigenic RSV F polypeptide is administered to a subject. This can increase the proportion of antibodies targeting epitopes specific to a particular conformation of F (e.g., antibodies targeting site Φ). Since F has a pre-fusion conformation in viruses that have not yet entered cells, an increased proportion of antibodies targeting pre-F can provide a greater degree of neutralization (e.g., expressed as a neutralization-to-binding ratio as described herein). Blocking can be achieved by engineering bulky moieties, such as N-glycans, in the vicinity of the shared epitope. For example, an N-glycosylation site not present in wild-type F can be added, for example, by mutating the appropriate residue to asparagine. In some embodiments, the blocked epitope is an epitope of antigenic site I of RSV F. In some embodiments, two or more epitopes shared between pre-F and post-F are blocked. In some embodiments, two or more epitopes of antigenic site I of RSV F are blocked. In some embodiments, one or more or all epitopes that topologically overlap with a blocked epitope are also blocked, and optionally, the blocked epitope is an epitope of antigenic site I of RSV F.

[0127] In some embodiments, the RSV F polypeptide comprises an asparagine substitution at one or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO: 1 (i.e., E328N, S348N, or R507N). In some embodiments, the RSV F polypeptide comprises an asparagine substitution at two or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO: 1 (i.e., E328N, S348N, or R507N). In some embodiments, the RSV F polypeptide comprises an asparagine substitution at positions 328, 348, and 507 of SEQ ID NO: 1 (i.e., E328N, S348N, and R507N).

[0128] As previously shown, it has been found that such asparagines can function as glycosylation sites (see WO 2019 / 195291, incorporated herein by reference). Additionally, without wishing to be bound by any particular theory, glycans at these sites may inhibit the development of antibodies against nearby epitopes, including epitopes common to pre-fusion and post-fusion RSV F proteins, when an RNA (e.g., mRNA) encoding an antigenic RSV F polypeptide is administered to a subject. In some embodiments, glycosylation of the asparagine corresponding to positions 328, 348, or 507 of SEQ ID NO: 1 blocks at least one epitope shared between pre-fusion RSV F and post-fusion RSV F, such as the epitope of antigenic site 1. Inhibiting the development of antibodies against epitopes common to pre-fusion and post-fusion RSV F proteins can be beneficial because it can direct the development of antibodies against epitopes specific to the pre-fusion RSV F protein, such as the site Φ epitope, which may have more effective neutralizing activity than antibodies against other RSV F epitopes. The site Φ epitope includes amino acid residues 62-69 and 196-209 of SEQ ID NO: 1. Thus, in some embodiments, the RSV F polypeptide includes amino acid residues 62-69 and 196-209 of SEQ ID NO: 1.

[0129] The RSV F polypeptides described herein may have deletions or substitutions of different lengths compared to wild-type RSV F. For example, in the RSV F polypeptide of SEQ ID NO: 1, positions 98-144 of the wild-type sequence (SEQ ID NO: 1) are replaced with GSGNVGL (SEQ ID NO: 15), resulting in a net deletion of 40 amino acids, such that positions 328, 348, or 507 of SEQ ID NO: 1 correspond to positions 288, 308, and 467 of SEQ ID NO: 3. Alternatively, in the RSV F polypeptide of SEQ ID NO: 3, positions 98-146 of the wild-type sequence (SEQ ID NO: 1) are replaced with GSGNVGLGG (SEQ ID NO: 16, positions 98-106 of SEQ ID NO: 3), resulting in a net deletion of 40 amino acids, such that positions 328, 348, or 507 of SEQ ID NO: 1 correspond to positions 290, 310, and 469 of SEQ ID NO: 3.

[0130] In general, positions in the constructs described herein can be mapped to the wild-type sequence of SEQ ID NO: 1 by pairwise alignment, for example using the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, gap penalty 10, gap extension penalty 0.5). See also the description of structural alignment provided herein as an alternative approach to identify corresponding positions.

[0131] In some embodiments, the RSV F polypeptide includes a mutation that adds a glycan to block an epitope on a pre-fusion antigen that is structurally similar to an epitope on the surface of post-fusion RSV F. In some embodiments, the glycan is added to specifically block an epitope that may be present in the post-fusion conformation of RSV F. In some embodiments, a glycan is added that blocks an epitope that may be present in the post-fusion conformation of RSV F but does not affect one or more epitopes present in the pre-fusion conformation of RSV F, such as the site Φ epitope.

[0132] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:2.

[0133] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:3.

[0134] In some embodiments, the RSV F polypeptide comprises a DS-CAV1 amino acid substitution (e.g., as described in McLellan et al., Science, 342(6158):592-598, 2013) with further modifications including at least one, two or three of the above asparagines. The CAV1 mutations are S190F and V207L ​​compared to SEQ ID NO:1. The DS mutations are S155C and S290C relative to SEQ ID NO:1.

[0135] In some embodiments, the amino acid substitution or pair of amino acid substitutions is an interprotomer stabilizing substitution. Exemplary substitutions that may be interprotomer stabilizing are V207L, N228F; I217V and E218F; I221L and E222M; or Q224A and Q225L, using the position numbering of SEQ ID NO:1.

[0136] In some embodiments, the amino acid substitution or pair of amino acid substitutions is intraprotomer stabilizing. Exemplary substitutions that may be intraprotomer stabilizing are V220I; and A74L and Q81L (using the position numbering of SEQ ID NO:1).

[0137] In some embodiments, the amino acid substitutions are predicted to be helix stabilizing, i.e., stabilize the helical domain of RSV F. Stabilization of the helical domain may generally contribute to the stability of the site Φ epitope and pre-fusion conformation of RSV F. Exemplary substitutions that may be helix stabilizing are N216P or I217P, using the position numbering of SEQ ID NO:1. Position 217 of SEQ ID NO:1 corresponds to position 177 of SEQ ID NO:3.

[0138] In some embodiments, the amino acid substitution is helix capping. In some embodiments, the amino acid substitution is helix PRO capping. Helix capping is based on the biophysical observation that mutation of a proline residue located in an alpha helix can disrupt helix formation, but a proline at the N-terminus of a helical region can help induce helix formation by stabilizing the PHI / PSI binding angle. Exemplary substitutions that can be helix capping are N216P or I217P, using the position numbering of SEQ ID NO:1.

[0139] In some embodiments, the amino acid substitution replaces the disulfide mutation of DS-CAV1. In some embodiments, the engineered disulfide of DS-CAV1 is returned to the wild type (C69S and / or C212S mutation of DS-CAV1 using the position numbering of SEQ ID NO: 1). In some embodiments, one or more C residues of DS-CAV1 are replaced with S residues to eliminate disulfide bonds. In some embodiments, the C69S or C212S substitution using the position numbering of SEQ ID NO: 1 eliminates disulfide bonds. In some embodiments, the RSV F polypeptide includes both C69S and C212S using the position numbering of SEQ ID NO: 1. In some embodiments, the reduction of the RSV F polypeptide (i.e., acceptance of electrons from a reducing agent) is blocked by replacing such cysteines, thereby eliminating disulfide bonds. In some embodiments, an I217P substitution using the position numbering of SEQ ID NO: 1 is included in the antigen in place of a substitution at C69 and / or C212.

[0140] In some aspects, the amino acid substitutions prevent proteolysis by trypsin or trypsin-like proteases. In some embodiments, such proteolytically preventing amino acid substitutions are within the heptad repeat region B (HRB) region of RSV F.

[0141] The appearance of fragments consistent with proteolysis of the RSV F polypeptide containing the wild-type HRB region suggested that lysine or arginine in this region is the target of proteolysis. Amino acid substitutions to remove K or R residues can be referred to as knockouts (KO). In some embodiments, K or R is replaced with L or Q. In some embodiments, K is replaced with L or Q. In some embodiments, the RSV F polypeptide comprises K498L and / or K508Q using the position numbering of SEQ ID NO: 1. The corresponding positions in SEQ ID NO: 3 are 458 and 468, respectively. In some embodiments, the RSV F polypeptide comprises both K498L and K508Q.

[0142] In some embodiments, the amino acid substitutions add glycans. In some embodiments, the amino acid substitutions increase glycosylation by adding glycans to the RSV F polypeptide. Substitutions that add glycans can also be referred to as engineered glycosylation, as compared to native glycosylation (no additional glycans).

[0143] In some embodiments, the amino acid substitution to add a glycan is a substitution with N. In some embodiments, the amino acid substitution with N allows for N-linked glycosylation. In some embodiments, the substitution with N is accompanied by a substitution with T or S at the second amino acid position C-terminal to N, forming an NxT / S glycosylation motif. In some embodiments, N is surface exposed.

[0144] Each of the above substitutions and mutations in the RSV F polypeptide is described in more detail in International Publication No. WO 2019 / 195291, which is incorporated herein by reference.

[0145] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a RSV F protein antigen, wherein the RSV F protein antigen comprises one or more of the following substitutions relative to the amino acid sequence set forth in SEQ ID NO:1: 1) amino acid positions 98 to 146 of SEQ ID NO:1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO:16); 2) amino acid substitutions S190F and V207L; 3) the amino acid substitution I217P; 4) amino acid substitutions E328N, S348N and R507N; 5) amino acid substitution L373R; 6) the amino acid substitution K498L; and 7) The amino acid substitution K508Q.

[0146] In another aspect, the present disclosure provides a RSV vaccine comprising an mRNA including an ORF encoding a RSV F protein antigen, wherein the RSV F protein antigen includes each of the following substitutions relative to the amino acid sequence set forth in SEQ ID NO:1: 1) amino acid positions 98 to 146 of SEQ ID NO:1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO:16); 2) amino acid substitutions S190F and V207L; 3) the amino acid substitution I217P; 4) amino acid substitutions E328N, S348N and R507N; 5) amino acid substitution L373R; 6) the amino acid substitution K498L; and 7) The amino acid substitution K508Q.

[0147] In a specific embodiment, the RSV F protein antigen comprises the transmembrane domain and cytoplasmic tail amino acid sequence of IMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 17).

[0148] In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a nucleic acid sequence set forth in any one of SEQ ID NOs: 4-6.

[0149] In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a nucleic acid sequence set forth in any one of SEQ ID NOs: 12-14.

[0150] iv. Lipid Nanoparticles (LNPs) The LNPs of the present disclosure can include lipids from four categories: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids (e.g., cholesterol), and (iv) helper lipids.

[0151] A. Cationic lipids Ionizable lipids promote mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH, promoting efficient encapsulation of negatively charged mRNA drug substances. Exemplary cationic lipids are shown in Table 1 below.

[0152] [Table 1]

[0153] [Table 2]

[0154] [Table 3]

[0155]

Table 4

[0156] The cationic lipids were [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-non-2-en-1-yl) 9-((4 -(Dimethylamino)butanoyl)oxy)heptadecanedioate (L319);9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102);[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315);[3-(Dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13 dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17 dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-Ch ol);Tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl))tetrapropionic acid (306Oi10);Decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9);Ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B);1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexa(octyl) 9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); and combinations thereof.

[0157] In certain embodiments, the cationic lipid is biodegradable.

[0158] In various embodiments, the cationic lipid is not biodegradable.

[0159] In some embodiments, the cationic lipid is cleavable.

[0160] In certain embodiments, the cationic lipid is not cleavable.

[0161] Cationic lipids are described in further detail in Dong et al. (PNAS. 111;11:3955-60. 2014); Fenton et al. (Adv. Mater. 28:2939. 2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference.

[0162] B. PEGylated lipids PEGylated lipid components provide control of nanoparticle size and stability. The addition of such components can prevent complex aggregation, increase circulation life, and provide a means to increase the delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters 268(1):235-71990). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0163] Contemplated PEGylated lipids include C6-C PEG-Ceramides, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (For example, 8, C 10 , C 12 , C 14 , C 16 Or C 18Examples of PEGylated lipids include, but are not limited to, polyethylene glycols (PEGs) of up to 5 kDa length covalently attached to lipids having alkyl chains of up to 5 kDa length. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkyloxypropyl carbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0164] In certain embodiments, the PEG has a high molecular weight, for example, 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.

[0165] C. Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf ...), and other lipids that are suitable for use in the treatment of cancer. al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol esters ("ICE"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24 cholestadiene); lanosterol (8,24 lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24, 25 dihydrolanosterol); zymosterol (5α-cholest-8,24dien-3β-ol); lathosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24 methylene cholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0166] D. Helper lipids The helper lipid enhances the structural stability of the LNP and aids the LNP in endosomal escape, which improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid with fusogenic properties to enhance the uptake and release of the drug payload. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-oleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serinecholine (DOPS), 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-distearoylphosphatidylethanolamine (DSPE) and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0167] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0168] In various embodiments, the LNP comprises: (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0169] E. Molar ratio of lipid components The molar ratio of the above components is important for the effectiveness of the LNP in delivering mRNA. The molar ratio of cationic lipid, PEGylated lipid, cholesterol-based lipid and helper lipid is A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipid in the LNP to total lipid (i.e., A) is 35-55%, such as 35-50% (e.g., 38-42%, such as 40% or 45-50%). In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, such as 1.5%). In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50% (e.g., 27-30%, such as 28.5% or 38-43%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35% (e.g., 28-32%, such as 30%, or 8-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs have a molar ratio of cationic lipid to helper lipid of greater than 1.

[0170] In certain embodiments, the LNPs of the disclosure include cationic lipid in a molar ratio of 35% to 55% or 40% to 50% (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% or 55% cationic lipid); Polyethylene glycol (PEG) conjugated (PEGylated) lipids in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50% or 2.75% molar ratio of PEGylated lipid); Cholesterol-based lipids in a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., cholesterol-based lipids in a molar ratio of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%); and Helper lipid in a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% helper lipid). and all molar ratios are relative to the total lipid content of the LNP.

[0171] In certain embodiments, the LNP comprises a 40% molar ratio of cationic lipid, a 1.5% molar ratio of PEGylated lipid, a 28.5% molar ratio of cholesterol-based lipid, and a 30% molar ratio of helper lipid.

[0172] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0173] In various embodiments, the cholesterol-based lipid is cholesterol.

[0174] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0175] In certain embodiments, the LNPs comprise OF-02 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0176] In certain embodiments, the LNPs comprise cKK-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0177] In certain embodiments, the LNPs comprise GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0178] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0179] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0180] In certain embodiments, the LNPs comprise SM-102 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0181] In certain embodiments, the LNPs comprise ALC-0315 in a molar ratio of 35% to 55%; ALC-0159 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0182] In certain embodiments, the LNP comprises OF-02 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid A."

[0183] In certain embodiments, the LNP comprises 40% molar ratio of cKK-E10, 1.5% molar ratio of DMG-PEG2000, 28.5% molar ratio of cholesterol, and 30% molar ratio of DOPE. This LNP formulation is referred to herein as "Lipid B."

[0184] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid C."

[0185] In a particular embodiment, the LNP comprises 40% molar ratio of GL-HEPES-E3-E12-DS-4-E10, 1.5% molar ratio of DMG-PEG2000, 28.5% molar ratio of cholesterol, and 30% molar ratio of DOPE. This LNP formulation is referred to herein as "Lipid D."

[0186] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid E".

[0187] In certain embodiments, the LNPs comprise 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) at a molar ratio of 50%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0188] In certain embodiments, the LNPs comprise (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 9.4%; cholesterol in a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.6%.

[0189] In certain embodiments, the LNPs comprise (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 10%; cholesterol in a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.7%.

[0190] To calculate the actual amount of each lipid contained in the LNP formulation, first, the molar amount of the cationic lipid is determined based on the desired N / P ratio (where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP). Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0191] F. Buffers and Other Ingredients The nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants) and / or other pharma- ceutically acceptable excipients to stabilize the nucleic acids and / or LNPs (e.g., to extend the shelf life of a vaccine product), facilitate administration of the LNP pharmaceutical composition, and / or enhance in vivo expression of the nucleic acids. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0192] The LNP compositions of the present disclosure can be provided in frozen liquid form or lyophilized form. A variety of cryoprotectants can be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition comprises, for example, 5-30% (e.g., 10%) (w / v) trehalose. When formulated with a cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at -20°C to -80°C.

[0193] The LNP compositions can be provided to the patient in a buffered aqueous solution (if previously frozen, they can be thawed, or if previously lyophilized, they can be reconstituted in the buffered aqueous solution at the bedside). The buffered solution can be isotonic, e.g., suitable for intramuscular or intradermal injection. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0194] V. Process for Producing LNP Vaccines The LNPs of the present invention can be prepared by various techniques. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by dissolving the lipids in a suitable solvent, depositing the selected lipids on the inner wall of a suitable container or container, and then evaporating the solvent to leave a thin film on the inside of the container, or by spray drying. An aqueous phase can then be added to the vessel with a vortexing motion, which results in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. Additionally, unilamellar vesicles can be formed by detergent removal techniques.

[0195] Various methods are described in US Patent Publication Nos. 2011 / 0244026, 2016 / 0038432, 2018 / 0153822, 2018 / 0125989, and 2021 / 0046192 and can be used to make LNP vaccines. One exemplary process involves encapsulating the mRNA by mixing with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in US Patent Publication No. 2016 / 0038432. Another exemplary process involves encapsulating the mRNA by mixing preformed LNPs with the mRNA, as described in US Patent Publication No. 2018 / 0153822.

[0196] In some embodiments, the process of preparing mRNA-loaded LNPs comprises heating one or more solutions to a temperature above ambient temperature, where one or more solutions are solutions containing preformed lipid nanoparticles, the solution containing mRNA, and the mixed solution containing the LNP-encapsulated mRNA. In some embodiments, the process comprises heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step. In some embodiments, the process comprises heating one or more of the solution containing the preformed LNP, the solution containing mRNA, and the solution containing the LNP-encapsulated mRNA during the mixing step. In some embodiments, the process comprises heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions are heated is greater than or equal to about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature is about 65° C.

[0197] Various methods may be used to prepare an mRNA solution suitable for the present disclosure. In some embodiments, the mRNA may be dissolved directly in a buffer solution as described herein. In some embodiments, the mRNA solution may be made by mixing the mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, the mRNA solution may be made by mixing the mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or a buffer at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or more.

[0198] In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a faster rate than the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or greater.

[0199] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or greater.

[0200] The process of incorporating desired mRNA into lipid nanoparticles is called "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312: 255-8. The nucleic acid incorporated into LNPs can be located completely or partially within the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or on the outer surface of the lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as "encapsulation", and the nucleic acid is completely or substantially contained within the internal space of the lipid nanoparticle.

[0201] Suitable LNPs can be made in a variety of sizes.In some embodiments, the reduction in size of lipid nanoparticles is associated with more efficient delivery of mRNA.Selection of suitable LNP size can take into account the target cell or tissue site and the application for which lipid nanoparticles are made.

[0202] Various methods are available for sizing the population of lipid nanoparticles. In various embodiments, the methods herein utilize a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is placed in a cuvette and then placed in the Zetasizer. The z-average diameter (nm) or cumulant average is considered to be the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) evaluation to guide efficient lipid nanoparticle synthesis.

[0203] In some embodiments, the majority of the purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the LNPs, have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm or about 80 nm). In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0204] In certain embodiments, the LNPs have an average diameter between 30 and 200 nm.

[0205] In various embodiments, the LNPs have an average diameter of 80-150 nm.

[0206] In some embodiments, the LNPs in the composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0207] In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99% of the LNPs in the composition have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm) or about 50-70 nm (e.g., about 55-65 nm) and are suitable for pulmonary delivery via nebulization.

[0208] In some embodiments, the dispersity or molecular size heterogeneity measure (PDI) of the LNPs in the pharmaceutical compositions provided by the present disclosure is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI can be measured by a Zetasizer machine as described above.

[0209] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50%-99% or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%).

[0210] In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio of greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In certain embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0211] In some embodiments, a pharmaceutical composition according to the present disclosure contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 μg-1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA.

[0212] In some embodiments, mRNA can be produced by chemical synthesis of DNA template or in vitro transcription (IVT). An exemplary process for producing and purifying mRNA is described in Example 1. In this process, the IVT process, a cDNA template is used to generate mRNA transcripts, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is purified again by depth filtration and TFF.

[0213] The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate or succinate buffer and can have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0 or about 6.5. The buffer can include other components such as salts (e.g., sodium salts, potassium salts and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, pH 4.5.

[0214] An exemplary, non-limiting process for making mRNA-LNP compositions includes mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled, homogenous manner, with the lipid:mRNA ratio being maintained throughout the mixing process. In this exemplary example, the mRNA is present in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanolic lipid solution are mixed in a 4:1 volume ratio in a "T" mixer equipped with an approximately "pulseless" pump system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. TFF can be used to concentrate and buffer exchange the nascent LNPs obtained immediately after formation by the T mixing process. The diafiltration process is a continuous operation in which the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow.

[0215] vi. Packaging and Use of mRNA-LNP RSV Vaccine The mRNA-LNP vaccines can be formulated or packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or nasopharyngeal (e.g., intranasal) administration. In various embodiments, the mRNA-LNP vaccines can be formulated or packaged for pulmonary administration. In various embodiments, the mRNA-LNP vaccines can be formulated or packaged for intravenous administration. The vaccine composition can be in the form of an extemporaneous preparation, where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately prior to use. The vaccine composition can also be shipped and provided in the form of an aqueous or frozen aqueous solution, and can be administered directly to a subject without reconstitution (after thawing, if previously frozen).

[0216] Thus, the present disclosure provides articles of manufacture such as kits that provide an mRNA-LNP vaccine in a single container, or that provide an mRNA-LNP vaccine in one container (e.g., a first container) and a physiological buffer for reconstitution in another container (e.g., a second container). The containers may contain single-use or multiple-use dosages. The containers may be pre-processed glass vials or ampoules. The articles of manufacture may also include instructions for use.

[0217] In certain embodiments, the mRNA-LNP vaccine is provided for use in intramuscular (IM) injection. The vaccine can be injected into the subject, for example, in the deltoid muscle of the upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for use in inhalation and is provided in a pre-filled pump, aerosolizer, or inhaler.

[0218] The mRNA-LNP vaccine can be administered to a subject in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against the target pathogen for a sufficient time (e.g., 1 year, 2 years, 5 years, 10 years, or lifelong). Sufficient immune protection can be, for example, prevention or alleviation of symptoms associated with infection by a pathogen. In some embodiments, to achieve a desired prophylactic effect, multiple doses (e.g., 2 doses) of the vaccine are administered (e.g., injection) to a subject in need thereof. The doses (e.g., a primary dose and a booster dose) can be separated by at least, for example, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, or 10 years.

[0219] VII. Vector In one aspect, vectors comprising the mRNA compositions disclosed herein are disclosed herein. RNA sequences encoding proteins of interest (e.g., mRNA encoding RSV F protein) can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0220] In certain embodiments, the vector can be used to express mRNA in host cells. In various embodiments, the vector can be used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is described in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0221] In some embodiments, the vectors disclosed herein may include, from 5' to 3', at least an RNA polymerase promoter; a polynucleotide sequence encoding a 5'UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3'UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may include a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0222] Various RNA polymerase promoters are known. In some embodiments, the promoter may be a T7 RNA polymerase promoter. Other useful promoters may include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3 and SP6 promoters are known.

[0223] Also disclosed herein are host cells (eg, mammalian cells, eg, human cells) that contain the vectors or RNA compositions disclosed herein.

[0224] Polynucleotides can be introduced into target cells using any of a number of different methods, including, but not limited to, commercially available methods, such as electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, a "gene gun" (see, e.g., Nishikawa, et al. (2001) Hum Gene Ther. 12(8):861-70), or biological particle delivery systems such as the TransIT-RNA transfection kit (Mirus, Madison, Wis.).

[0225] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0226] Regardless of the method used to introduce exogenous nucleic acid into a host cell or otherwise expose the cell to an inhibitor of the present disclosure, a variety of assays can be performed to confirm the presence of the mRNA sequence in the host cell.

[0227] VIII. Self-replicating and trans-replicating RNA Self-replicating RNA: In one embodiment, a self-replicating RNA encoding a RSV F protein is disclosed herein.

[0228] Self-replicating RNA can be produced, for example, by using replication elements derived from alphaviruses to replace structural viral proteins with nucleotide sequences encoding a protein of interest (e.g., RSV F protein). Self-replicating RNAs are typically positive-stranded molecules that can be directly translated after delivery to a cell, which translation then provides an RNA-dependent RNA polymerase that generates both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs and collinear subgenomic transcripts can be translated themselves to provide in situ expression of the encoded antigen (i.e., RSV F protein antigen), or can be transcribed to provide additional transcripts with the same sense as the delivered RNA that are translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is a large amplification of the number of introduced replicon RNAs, such that the encoded antigen becomes the major polypeptide product of the cell.

[0229] One suitable system for achieving self-replication in this way is to use alphavirus-based replicons. These replicons are positive-stranded (positive sense) RNAs that result in the translation of a replicase (or replicase transcriptase) after delivery to the cell. The replicase is translated as a polyprotein that self-cleaves to provide a replication complex that generates a genomic strand copy of the plus-stranded delivered RNA. These negative (-) strand transcripts can themselves be transcribed to give further copies of the positive strand parent RNA and can also give subgenomic transcripts that code for antigens. Translation of the subgenomic transcripts thus results in in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and the like. Mutant or wild-type viral sequences can be used, for example, the attenuated TC83 mutant of VEEV has been used in the replicon. See the following references: WO 2005 / 113782, incorporated herein by reference.

[0230] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an RSV F protein antigen. The polymerase may be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Although naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, a self-replicating RNA may result in the production of its own genomic RNA copy in a cell, but not in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot persist in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are absent in the self-replicating RNA of the present disclosure, and their place is taken by a gene encoding an immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion protein. Self-replicating RNA is described in further detail in WO2011005799, which is incorporated herein by reference.

[0231] Trans-replicating RNA: In one embodiment, a trans-replicating RNA encoding a RSV F protein is disclosed herein.

[0232] Trans-replicating RNA has similar elements to the self-replicating RNA described above. However, in trans-replicating RNA, two separate RNA molecules are used. The first RNA molecule encodes the RNA replicase described above (e.g., alphavirus replicase), and the second RNA molecule encodes a protein of interest (e.g., RSV F protein antigen). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby greatly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNA is described in more detail in WO2017162265, which is incorporated herein by reference.

[0233] IX. Pharmaceutical Compositions RNA purified according to the present disclosure may be useful as a component in pharmaceutical compositions for use, for example, as a vaccine. These compositions typically include RNA and a pharma- ceutically acceptable carrier. The pharmaceutical compositions of the present disclosure may also include one or more additional components, such as a small molecule immunostimulant (e.g., a TLR agonist). The pharmaceutical compositions of the present disclosure may also include a delivery system for RNA, such as a liposome, an oil-in-water emulsion, or a microparticle. In some embodiments, the pharmaceutical composition includes a lipid nanoparticle (LNP). In certain embodiments, the composition includes an antigen-encoding nucleic acid molecule encapsulated within the LNP.

[0234] X. Vaccination Methods The RSV vaccines disclosed herein can be administered to a subject to induce an immune response against the RSV F protein, and the subject's anti-antigen antibody titer is increased after vaccination compared to the anti-antigen antibody titer of a subject not vaccinated with the RSV vaccine disclosed herein or compared to an alternative vaccine against RSV. An "anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0235] In one aspect, the present disclosure provides a method of inducing an immune response against RSV or protecting a subject against RSV infection, comprising administering to a subject a RSV vaccine as described herein. The present disclosure also provides a RSV vaccine as described herein for use in inducing an immune response against RSV or protecting a subject against RSV infection. The present disclosure also provides a RSV mRNA as described herein for use in producing a vaccine for inducing an immune response against RSV or protecting a subject against RSV infection.

[0236] In a specific embodiment, the subject has a higher serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:1.

[0237] In certain embodiments, a subject has a comparable serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a RSV protein vaccine co-administered with an adjuvant.

[0238] In a specific embodiment, the RSV vaccine increases the serum concentration of antibodies having binding specificity for site Φ of the RSV F protein.

[0239] In certain embodiments, the subject has a lower serum concentration of antibodies having binding specificity for site I or site II of the RSV F protein after administration of the RSV vaccine compared to a subject administered a RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:2.

[0240] In certain embodiments, the RSV vaccine increases serum concentrations of neutralizing antibodies in subjects with pre-existing RSV immunity.

[0241] In order that the invention may be better understood, the following examples are set forth, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. EXAMPLES

[0242] The foregoing description of specific embodiments fully reveals the general nature of the present disclosure, so that others, by applying knowledge within the skill of those of ordinary skill in the art, can easily modify and / or adapt such specific embodiments to various applications without departing from the general concept of the present disclosure and without undue experimentation. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the expressions or terms used herein are intended to be descriptive rather than limiting, as the terms or terms used herein would be interpreted by those of ordinary skill in the art in light of the teaching and guidance.

[0243] Example 1: mRNA encoding the RSV F protein Three different RSV F proteins were selected for testing as mRNA-based vaccines. The F protein designated FD1 corresponds to the WT RSV F protein. The F protein designated FD2 corresponds to a soluble RSV F protein that lacks the transmembrane domain and cytoplasmic tail and contains a C-terminal fibritin trimerization domain (also known as the T4 foldon). The F protein designated FD3 corresponds to the pre-fusion RSV F protein. The amino acid sequences of each RSV F protein are listed below.

[0244] FD1: [ka]

[0245] FD2: [ka]

[0246] FD3: [ka]

[0247] The mRNA described herein includes an open reading frame (ORF) encoding a RSV F protein antigen, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR) and at least one polyadenylation (poly(A)) sequence. The mRNA has the following structure: [ka] The 5' cap further comprises the following:

[0248] The nucleic acid sequences of each of the mRNA open reading frames (ORFs) encoding the RSV F protein are listed below.

[0249] FD1 mRNA ORF: [ka]

[0250] FD2 mRNA ORF: [ka]

[0251] FD3 mRNA ORF: [ka]

[0252] The nucleic acid sequence of each DNA template encoding the RSV F protein is listed below.

[0253] FD1 DNA: [ka]

[0254] FD2 DNA: [ka]

[0255] FD3 DNA: [ka]

[0256] The nucleic acid sequences of the 5'UTR and 3'UTR are listed below.

[0257] 5'UTR: [ka]

[0258] 3'UTR: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 11)

[0259] The nucleic acid sequences of each of the full-length mRNAs encoding the RSV F protein are listed below.

[0260] FD1 mRNA: [ka]

[0261] FD2 mRNA: [ka]

[0262] FD3 mRNA: [ka]

[0263] RSV F mRNA Protein Expression: Protein expression of FD1, FD2 and FD3 mRNA constructs was assessed. The mRNA constructs were transfected into human embryonic kidney cells (HEK) and after 24 hours cell lysates or supernatants were harvested for analysis by Western blot. Figure 1A shows a Western blot image of protein harvested from transfected cells and detected using the 5353C75 mouse monoclonal antibody. The FD1 construct produced a moderately intense band at the expected molecular weight (MW) of approximately 50 kDa based on the DS-Cav1 protein control, but there was also a very dense band at <30 kDa. The FD2 band was expressed at the correct MW but was substantially less dense and showed lower expression than the 50 kDa band of FD1. The FD3 band was much larger and smeared from 60-80 kDa, which is consistent with FD3 being a glycosylated protein. Neither FD2 nor FD3 expressed protein bands below 30 kDa as did FD1. To investigate the lower molecular weight band observed with FD1, in vitro translation was performed on the mRNA. As shown in Figure 1B, a Western blot was prepared using HEK transfected proteins derived from FD1 mRNA in the lane marked "FD1-T" and in vitro translated products derived from FD1 mRNA in the lane marked "FD1-IVT." The lack of expression of the small MW band in the FD1-IVT lane of this blot indicates that the small MW band may result from a problem with protein processing by HEK cells.

[0264] To further investigate low molecular weight band expression by FD1 and ensure consistency between mRNA lots, two newly transcribed mRNA lots were prepared for all three FD constructs. These mRNAs were transfected into HEK cells and nucleofected into human skeletal muscle cells (HSkMCs) as shown in Figures 2A and 2B.

[0265] Finally, immunostaining was performed on membrane-bound FD1 and FD3 constructs to assess cell surface expression and binding of monoclonal antibodies to known antigenic sites. HEK cells were transfected with FD1 or FD3 mRNA and probed with monoclonal antibodies 24 hours after transfection. As shown in Figure 3, both constructs had high signals when probed with site II-specific monoclonal antibodies, but unlike FD3-transfected cells, the FD1 construct had little signal above background when probed with site φ-specific monoclonal antibody D25 (mock transfected). This indicates that the RSV-F protein expressed on the surface of FD1-transfected cells does not present the antigenic site with the highest neutralization potential. Together with western blots showing unexpected banding patterns (e.g., Figures 1A, 1B, 2A, and 2B), these data suggest that FD1 may not induce effective protection in vivo and immunogenicity studies should be performed to confirm this.

[0266] Example 2: Immunogenicity of mRNA encoding RSV F protein in mice The immunogenicity of mRNAs encoding the above RSV F proteins FD1, FD2 and FD3 was tested in non-immunized (naive) mice. Each mRNA was encapsulated in lipid nanoparticles (LNPs).

[0267] Each LNP-mRNA composition was administered to naive mice at a dose of 1 μg / mouse. As a control, pre-fusion F protein nanoparticles (Pre-F-NPs) were administered to mice with alum-85 adjuvant. Alum-85 (also known as alhydrogel-85) is a type of aluminum hydroxide-based adjuvant. Pre-F-NPs utilize a ferritin domain at the C-terminus of the Pre-F protein, where each ferritin domain self-assembles with other ferritin domains to form spherical nanoparticles. Serum was extracted from immunized mice on days 0, 21, and 35 after injection. RSV F protein antibody titers (Figure 9A) and RSV microneutralization titers (Figure 9B) were measured. Antibody titers were measured as follows: ELISA plates were coated with 1 μg / ml of RSV pre-fusion F protein. After blocking, the plates were then subjected to serial dilutions of each serum sample covering a dilution range of 1:1000 to 1:729,000. Plates were then detected using anti-mouse secondary antibody conjugated to horseradish peroxidase, followed by visualization using Pierce 1-Step Ultra TMB-ELISA Substrate Solution. Plates were then read at 450 nm on a SpectraMax plate reader. Titers were reported as the highest serum dilution that resulted in an optical density of greater than 0.2.

[0268] RSV microneutralization titers were measured as follows: Vero cells (ATCC CCL-81) were seeded at 30,000 cells / well in 96-well plates suitable for fluorescence reading one day prior to infection. Serum samples were heat inactivated and serially diluted 4-fold from 1:20 to 1:81,920. The diluted serum was combined 1:1 with RSV strain A2 expressing a green fluorescent protein reporter and incubated for 1 hour. The serum-virus mixture was added to the cell plate, which was incubated for 24 hours. The plate was then read on a high content imager and the fluorescent events were quantified. Serum 50% neutralization titers were calculated using a 4-parameter logistic regression in SoftMax GxP.

[0269] Among FD1, FD2 and FD3 mRNAs, the data show that FD1 and FD3 mRNAs induced the highest RSV F protein binding antibody titers and FD2 and FD3 mRNAs induced the highest RSV neutralization in mice.

[0270] Example 3: Immunogenicity of mRNA encoding the RSV F protein in the modular immune in vitro construct (MIMIC®) system The MIMIC® system uses circulating immune cells of individual donors to recapitulate individual human immune responses. Human exposure history to RSV is represented in immune populations of human donor cells used to develop the MIMIC® B-cell Lymphoid Tissue Equivalent (LTE) module. This module allows for the measurement of "recall" responses in circulating lymphocytes, a situation expected during immunization of human populations with pre-existing immunity to RSV. The MIMIC® system is further described in Higbee et al. (Altern. Lab Anim. 37: Suppl 1: 19-27. 2009), which is incorporated herein by reference. The mRNA-LNP composition was incubated with the MIMIC® system. Each LNP was composed of 40% cationic lipid F, 30% phospholipid DOPE, 1.5% PEGylated lipid DMG PEG2000, and 28.5% cholesterol.

[0271] To confirm antibody responses to FD1, FD2, and FD3, a dose of 0.37 μg of mRNA-LNP was used in MIMIC® B-cell LTE. After 14 days, MIMIC® supernatants were collected and tested for antibody binding to Pre-F and Post-F in a Luminex-based Antibody Forensics assay.

[0272] Anti-pre-F protein IgG titers were measured for each of FD1, FD2 and FD3 mRNA, along with Pre-F NP as a control. As shown in Figure 10A, each of the three mRNAs stimulated strong production of anti-pre-F protein IgG. The anti-Pre-F / anti-Post-F antibody ratio was also measured. As shown in Figure 10B, FD3 mRNA stimulated anti-pre-F antibody production to a greater extent than FD1 or FD2 mRNA.

[0273] The neutralizing antibody titers induced by the above-mentioned stimulation were determined in the MIMIC® system. As shown in Figure 11, each of FD1, FD2, and FD3 mRNA stimulated high anti-RSV neutralizing titers.

[0274] Example 4: Immunogenicity of mRNA encoding the RSV F protein in non-human primates (NHPs) Immunogenicity experiments were performed in NHPs. Each mRNA was encapsulated in LNPs composed of 40% cationic lipid OF-02, 30% phospholipid DOPE, 1.5% PEGylated lipid DMG PEG2000, and 28.5% cholesterol ("Lipid A" LNP formulation).

[0275] Each LNP-mRNA composition was administered to naive NHPs at a dose of 10 μg / animal. As a control, Pre-F-NP was administered to NHPs with AF03 adjuvant. AF03 is a squalene-based emulsion adjuvant further described in Klucker et al. (J. Pharma. Scien. 101(12):4490-4500. 2012). Serum was extracted from immunized NHPs on days 0, 28, and 56 after injection. RSV F protein antibody titers (FIG. 4A) and RSV microneutralization titers (FIG. 4B) were measured. The data show that all three mRNAs induced high RSV F protein binding antibody titers, with FD2 and FD3 mRNAs inducing the highest RSV neutralization in NHPs.

[0276] Immunogenicity experiments were also performed in NHPs pre-immunized with Pre-F NPs. Each mRNA was encapsulated in the same LNPs used above in naive NHPs.

[0277] Anti-RSV F protein antibodies generated in immunized NHPs were further characterized by competitive ELISA. Serum from immunized NHPs was used to determine the ability of the sera to compete with three different anti-RSV F protein antibodies. Antibody D25 binds to site φ on the pre-F protein (McLellan et al., Science. 340(6136):1113-7. 2013). Antibody Synagis (palivizumab) binds to site II (Johnson et al. J. Infect. Dis. 176(5):1215-24. 1997). Antibody 131-2a binds to site I (Widjaja et al. J. Virol. 90(13):5965-5977. 2016). As shown in Figure 5, serum from NHPs immunized with FD3 mRNA or pre-F NPs adjuvanted showed high Log2IT by D25 antibody. 50 As demonstrated by the β-peptide values, most of the FD3-engineered F proteins had high site φ antibodies. In addition, limited site I (post-fusion F protein is preferred, 131-2a) or site II (Synagis) antibodies were produced. This may be due to modifications made to the F protein of the FD3 design, which promote the formation of pre-fusion F protein and introduced glycosylation sites, masking less productive epitopes. This allows immune refocusing to the most potent epitopes of site φ.

[0278] In contrast to FD3, FD2 mRNA induced similar levels of site φ, II and I specific antibodies. Similar results were observed in Espeseth et al. (npj Vaccines.5(1):16.2020). Site II antibodies are less potent than site φ antibodies. Furthermore, less effective antibodies from site I may affect neutralizing activity or enhance infection.

[0279] Pre-immune NHP assessment: Another factor to consider when comparing FD2 and FD3 is their ability to induce memory B cell recall responses in vaccinated individuals. Soluble proteins, such as those produced by the FD2 construct, are generally less efficient at cross-linking B cell receptors (BCRs) and activating memory B populations compared to membrane-bound proteins (Kowalski et al. Molecular Therapy. 27(4):710-728. 2019; Maruggi et al. Molecular Therapy. 27(4):757-772. 2019; Pardi et al. Nature Reviews Drug Discovery. 17(4):261-279. 2018). Vaccination with the FD2 construct resulted in strong RSV neutralizing antibody responses in naive populations of mice and monkeys, but the FD2 construct produces a soluble RSV-F protein and should therefore be tested in pre-immune populations to assess boosting responses. To test this, 12 monkeys previously vaccinated with RSV Pre-F NP with adjuvant were used and divided into two groups (n=6) based on pre-immune status (by RSV-F ELISA) and previous study participation. Animals selected for the pre-immune boosting study were pre-immunized with adjuvanted RSV Pre-F NP subunit vaccine. Briefly, three studies were selected whose last immunization date was at least 6 months before the start of the pre-immune boosting study. All eligible and available animals from these three studies were screened for RSV titer by ELISA, and 12 animals were selected for the pre-immune boosting study. Based on RSV titer, sex, and previous study participation, animals were placed into either the FD2 or FD3 boosting group.

[0280] One group was boosted with 5 μg of FD2 mRNA-LNP (Lipid A LNP formulation) and the other group was boosted with 5 μg of FD3 mRNA-LNP (Lipid A LNP formulation). Blood was collected at DO, D14 and D28 and serum was assessed by RSV-F ELISA and RSV MNA to determine the fold increase in binding and neutralizing antibodies, respectively. Both the binding antibody titers shown in FIG. 6A and the neutralizing antibody titers shown in FIG. 6B significantly increased from DO to D14 (p<0.001) for the FD2 and FD3 boost groups, but did not change between D14 and D28. No difference was observed between the responses induced by FD2 and FD3, indicating that both constructs can effectively boost pre-immune individuals.

[0281] Example 5: Cationic lipid screening The effect of different cationic lipids in LNPs was tested on LNP-encapsulated RSV F protein mRNA. Cationic lipids cKK-E10, OF-02, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14 and GL-HEPES-E3-E10-DS-3-E18-1 were tested. Each LNP was composed of 40% of one of the five cationic lipids, 30% phospholipid DOPE, 1.5% PEGylated lipid DMG-PEG2000 and 28.5% cholesterol. LNPs with cationic lipid MC3 were also used and considered the industry benchmark (Jayaraman et al. Angew Chem Int Ed. 51:8529-33. 2012).

[0282] The characteristics of the LNPs tested are shown in Table 2 below.

[0283] [Table 5]

[0284] NHPs were administered the LNP-FD3 mRNA composition. Groups of six cynomolgus monkeys were administered a 5 μg dose of mRNA encapsulated in the LNPs or a 10 μg dose of the RSV Pre-F NP subunit control vaccine adjuvanted with Al(OH)3 by intramuscular (IM) injection on DO and D21. Monkeys were bled before each vaccine administration and 2 weeks after the last vaccination (D35). As shown in FIG. 7, all cationic lipids tested effectively induced the production of anti-RSV F protein antibodies to a level similar to that of Pre-F NPs with aluminum adjuvant.

[0285] As shown in FIG. 8, all cationic lipids tested produced efficacious RSV neutralization titers to levels similar to those of Pre-F NP with aluminum adjuvant.

[0286] The cumulative results of Figures 7 and 8 are shown in Tables 3 and 4 below.

[0287] [Table 6]

[0288] [Table 7]

[0289] A better quality immune response is demonstrated by a lower value for the antibody titer / neutralization titer ratio, where LNPs containing cationic lipid cKK-E10 showed the best quality immune response, but all cationic lipids showed superior quality immune responses compared to the non-mRNA vaccine Pre-F NP.

[0290] Example 6: A Phase I / II Randomized, Double-Blind, Placebo-Controlled, Multi-Arm Dose-Ranging Study to Evaluate the Safety and Immunogenicity of RSV mRNA Vaccine Candidates Formulated with Either LNP cKK-E10 or LNP GL-HEPES-E3-E12-DS-4-E10 in Adult Participants Aged 18-50 or 60 and Older Reasons for the test The clinical trial described herein will test the safety and immunogenicity of a RSV mRNA LNP vaccine. The RSV mRNA LNP vaccine contains RSV mRNA in one of two encapsulated LNP formulations (i.e., LNPs containing either cKK-E10 or GL-HEPES-E3-E12-DS-4-E10) administered at three different doses (i.e., low, medium, or high) to healthy adults aged 18-50 years in the sentinel cohort and 60 years or older in the primary and booster cohorts. The RSV mRNA LNP vaccine will be provided as a liquid frozen solution in vials for intramuscular (IM) administration.

[0291] Quick Overview The objective of this study is to evaluate the safety and immunogenicity of a single intramuscular (IM) injection of three dose levels of a respiratory syncytial virus (RSV) messenger ribonucleic acid (mRNA) vaccine candidate formulated with two different lipid nanoparticles (LNPs) (i.e., LNPs containing cKK-E10 or GL-HEPES-E3-E12-DS-4-E10) in healthy adult participants aged 18-50 years in the sentinel cohort and 60 years or older in the primary and booster cohorts. The primary objective of this study is to evaluate the safety and immunogenicity profile across dose level groups (low, medium and high doses) with the two LNPs (cKK-E10 and GL-HEPES-E3-E12-DS-4-E10). Additionally, the study will evaluate the safety and immunogenicity of a booster vaccination administered 12 months after the primary vaccination in a subset of the study population. The duration of participation for each participant was 12 months in the sentinel and main cohorts, and 24 months overall for the subset of participants enrolled in the booster cohort.

[0292] [Table 8]

[0293] the purpose Primary Objective: The primary objective is to evaluate the safety and immunogenicity profile of three different dose levels (i.e., low, medium and high doses) of the RSV mRNA vaccine described herein encapsulated in either LNPs containing cKK-E10 or LNPs containing GL-HEPES-E3-E12-DS-4-E10.

[0294] Secondary Objectives: The secondary objectives are to evaluate: (1) the safety profile of a booster vaccination given 12 months after primary vaccination in a subset of participants; (2) the durability of the immune response at 3, 6, and 12 months after primary vaccination; (3) the durability of the immune response after a booster vaccination 12 months after primary vaccination in a subset of participants.

[0295] Primary and secondary endpoints were established to test whether the primary and secondary objectives were met. Tables 6 and 7 below summarize the description and assessment timeframes of the primary and secondary endpoints, respectively.

[0296] [Table 9]

[0297] [Table 10]

[0298] Study population Inclusion and Exclusion Criteria Inclusion Criteria Sentinel cohort aged 18-50 years on the date of inclusion, primary cohort and booster cohort aged 60 years or older on the date of inclusion. Female participants were eligible to participate if they were not pregnant or lactating and of non-fertile potential. To be considered of non-fertile potential, women must be postmenopausal for at least 1 year or surgically sterile. Attend all scheduled visits and be able to comply with all study procedures. Signed and dated informed consent form.

[0299] Exclusion Criteria Participants were excluded from the study if any of the following criteria applied: known or suspected congenital or acquired immune deficiency; receiving immunosuppressive therapy such as anticancer chemotherapy or radiation therapy within 6 months; or long-term systemic corticosteroid therapy (prednisone or equivalent for a period of more than 2 consecutive weeks within the past 3 months); known systemic hypersensitivity to any of the study intervention components (e.g., polyethylene glycol and polysorbate); history of a life-threatening reaction to the study intervention used in the study or to products containing any of the same substances; mRNA Any allergic reaction (e.g., anaphylaxis) after administration of the COVID-19 vaccine; history of RSV-related disease diagnosed clinically, serologically, or microbiologically within the past 12 months; history of myocarditis, pericarditis, and / or myocarditis; thrombocytopenia or bleeding disorder, contraindication to IM injection in the investigator's judgment; bleeding disorder or administration of anticoagulants within 3 weeks prior to administration, contraindication to intramuscular injection; chronic illness at any stage that, in the investigator's opinion, may interfere with the conduct or completion of the study; alcohol, prescription drug, or substance abuse that, in the investigator's opinion, may interfere with the conduct or completion of the study; receipt of any vaccine other than the mRNA vaccine in the 4 weeks prior to administration of any study intervention or within 4 weeks after administration of any study intervention. Planned receipt of any vaccine other than an mRNA vaccine; receipt of any mRNA vaccine in the 60 days prior to administration of any study intervention or planned administration of any mRNA vaccine in the 60 days after administration of any study intervention; previous vaccination against RSV with an investigational vaccine; receipt of immunoglobulin, blood or blood-derived products in the past 3 months; receiving oral or injectable antibiotic therapy within 72 hours prior to the first blood draw; participation at the time of study enrollment (or in the 4 weeks prior to administration of the first study intervention) or planned participation during the duration of this study in another clinical trial investigating a vaccine, drug, medical device or medical procedure; being deprived of liberty by administrative or court order or being in an emergency or involuntarily hospitalized;Self-reported or documented human immunodeficiency virus (HIV) detected by any FDA approved / validated test, Hepatitis B Virus Surface Antigen (HBsAg), Hepatitis B Core Antibody (HBcAb), Hepatitis C Virus Antibody (HCV Ab) or positive SARS-CoV-2 RTPCR or antigen test; or identified as an investigator or employee of the investigator or clinical trial center and directly involved in the proposed study, or identified as an immediate family member (i.e., parent, spouse, and natural or adopted child) of an investigator or employee and directly involved in the proposed study;

[0300] Duration per participant Each participant will participate for 12 months in the sentinel and primary cohorts, and 24 months overall for the subset of participants enrolled in the booster cohort. Participants in the sentinel cohort (one intramuscular (IM) injection) will be followed for 12 months after vaccination. Participants in the primary cohort (one IM injection) will be followed for 12 months after vaccination. Participants in the booster cohort (one IM injection 12 months after primary vaccination) will be followed for 12 months after administration of the booster dose.

[0301] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims.

[0302] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A respiratory syncytial virus (RSV) vaccine comprising messenger RNA (mRNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to or consisting of the amino acid sequence of SEQ ID NO:

3.

2. The RSV F protein antigen contains the following substitutions relative to the amino acid sequence set forth in SEQ ID NO:1: 1) amino acid positions 98-146 of SEQ ID NO:1 are replaced with the amino acid sequence GSGNVGLGG (SEQ ID NO:16); 2) amino acid substitutions S190F and V207L; 3) amino acid substitution I217P; 4) amino acid substitutions E328N, S348N, and R507N; 5) amino acid substitution L373R; 6) the amino acid substitution K498L; and 7) Amino acid substitution K508Q 2. The RSV vaccine of claim 1, comprising one or more of:

3. The RSV F protein antigen is a pre-fusion protein; the ORF is codon-optimized; the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR) and at least one polyadenylation (poly(A)) sequence; and / or The mRNA comprises at least one chemical modification, and optionally: (i) at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified; and / or (ii) at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80%, at least 85%, at least 90%, at least 95% or 100% are chemically modified; and / or (iii) the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine; The RSV vaccine of claim 1.

4. 10. The RSV vaccine of claim 1, wherein the mRNA is formulated in a lipid nanoparticle (LNP).

5. The LNPs comprise at least one cationic lipid, and optionally, The cationic lipid may or may not be biodegradable; the cationic lipid is cleavable or non-cleavable; and / or 5. The RSV vaccine of claim 4, wherein the cationic lipid is selected from the group consisting of GL-HEPES-E3-E12-DS-4-E10, OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, and GL-HEPES-E3-E12-DS-3-E14.

6. 5. The RSV vaccine of claim 4, wherein the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

7. The LNP is a molar ratio of 35% to 55%, optionally 40%, of a cationic lipid; polyethylene glycol (PEG) conjugated (PEGylated) lipids at a molar ratio of 0.25% to 2.75%, optionally 1.5%; a cholesterol-based lipid in a molar ratio of 20% to 45%, optionally 28.5%, and Helper lipid in a molar ratio of 5% to 35%, optionally 30% 5. The RSV vaccine of claim 4, comprising:

8. The PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); the cholesterol-based lipid is cholesterol; and / or The helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The RSV vaccine of claim 6.

9. The LNP is GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and 30% molar ratio of DOPE 5. The RSV vaccine of claim 4, comprising:

10. The LNP is cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and 30% molar ratio of DOPE 5. The RSV vaccine of claim 4, comprising:

11. 2. The RSV vaccine of claim 1, wherein the mRNA comprises a nucleic acid sequence having at least 80% identity to the nucleic acid sequence set forth in SEQ ID NO:

6.

12. 2. The RSV vaccine of claim 1, wherein the mRNA comprises a nucleic acid sequence having at least 80% identity to the nucleic acid sequence set forth in SEQ ID NO:

14.

13. The mRNA contains the following structural elements: (i) a molecule having the following structure: 【Chemistry 1】 a 5' cap having (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 11; and (v) poly(A) tail 2. The RSV vaccine of claim 1, comprising:

14. 1. A respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen, said mRNA comprising the following structural elements: (i) a molecule having the following structure: 【Chemistry 2】 a 5' cap having (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 11; and (v) poly(A) tail Including; The mRNA is GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and 30% molar ratio of DOPE A respiratory syncytial virus (RSV) vaccine formulated in lipid nanoparticles (LNPs) comprising:

15. 1. A respiratory syncytial virus (RSV) vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding an RSV F protein antigen, said mRNA comprising the following structural elements: (i) a molecule having the following structure: 【Transformation 3】 a 5' cap having (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 10; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 11; and (v) poly(A) tail Including; The mRNA is cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and 30% molar ratio of DOPE A respiratory syncytial virus (RSV) vaccine formulated in lipid nanoparticles (LNPs) comprising:

16. 16. An RSV vaccine for use in inducing an immune response against RSV or protecting a subject against RSV infection, comprising administering to a subject the RSV vaccine of any one of claims 1 to 15, Optionally, the subject has a higher serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject administered an RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO: 1; the subject has a comparable serum concentration of neutralizing antibodies to RSV after administration of the RSV vaccine compared to a subject receiving a protein RSV vaccine, optionally wherein the protein RSV vaccine is co-administered with an adjuvant; The RSV vaccine increases serum concentrations of antibodies having binding specificity for site Φ of the RSV F protein; the subject has a lower serum concentration of antibodies having binding specificity for Site I or Site II of the RSV F protein after administration of the RSV vaccine compared to a subject administered an RSV vaccine comprising an mRNA ORF encoding the RSV F protein antigen of SEQ ID NO:2; and / or The RSV vaccine increases serum concentrations of neutralizing antibodies in subjects with pre-existing RSV immunity.

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