Respiratory syncytial virus mRNA vaccine

Abstract

Provided herein are respiratory syncytial virus (RSV) vaccine compositions comprising a messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding an RSV F mutant B strain protein, and optionally an mRNA containing an ORF encoding an RSV F mutant A strain protein, and methods for inducing an immune response against RSV by administering an effective amount of the RSV vaccine composition to a subject in need thereof. Also provided herein are respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccine compositions comprising an mRNA containing an ORF encoding an RSV F mutant A strain protein, an mRNA containing an ORF encoding an RSV F mutant B strain protein, and an mRNA containing an ORF encoding an hMPV F protein, and methods for inducing an immune response against RSV and hMPV by administering an effective amount of the RSV and hMPV vaccine compositions to a subject in need thereof.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 483,099, filed February 3, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] [Incorporation by reference to sequence listing] The contents of the electronically submitted sequence listing submitted herewith, with file name: F293995_sequence listing as filed, size: 35079 bytes, and creation date: December 19, 2023, are incorporated herein by reference in their entirety.

[0003] Provided herein are respiratory syncytial virus (RSV) vaccine compositions comprising messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding an RSV F mutant B strain protein, and optionally an mRNA containing an ORF encoding an RSV F mutant A strain protein, and methods for inducing an immune response against RSV by administering an effective amount of the RSV vaccine composition to a subject in need thereof.

[0004] Also provided herein are respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccine compositions comprising an mRNA containing an ORF encoding an RSV F mutant A strain protein, an mRNA containing an ORF encoding an RSV F mutant B strain protein, and an mRNA containing an ORF encoding an hMPV F protein, as well as methods for inducing an immune response against RSV and hMPV by administering an effective amount of the RSV and hMPV vaccine compositions to a subject in need thereof. [Background technology]

[0005] Respiratory syncytial virus (RSV) is a common respiratory virus that infects the nose, throat, lungs, and airways. Although vaccine development is one of the best strategies for preventing RSV, no RSV vaccine is currently approved. An RSV vaccine is needed.

[0006] Human metapneumovirus (hMPV) is a similar but distinct virus. The seasonal epidemiology of hMPV is similar to that of RSV. Although vaccine development is one of the best strategies for preventing RSV and hMPV, no RSV or hMPV vaccines are currently licensed. RSV and hMPV vaccines are needed. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure provides a respiratory syncytial virus (RSV) vaccine composition comprising a messenger ribonucleic acid (mRNA) including an open reading frame (ORF) encoding a RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO: 1. In one embodiment, the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the mRNA including the ORF encoding the RSV F mutant B strain protein further includes a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the RSV F mutant B strain protein has the structure of 5' UTR-RSV F mutant B strain protein-encoding ORF-3' UTR-poly(A) tail, and the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the poly(A) tail has a length of 50 nucleotides to 250 nucleotides. In one embodiment, the mRNA having the structure of 5' UTR-RSV F mutant B strain protein-encoding ORF-3' UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 3. In another embodiment, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein has a nucleotide sequence having at least 80% identity to SEQ ID NO:3.

[0008] The present disclosure also provides a RSV vaccine composition comprising an mRNA including an ORF encoding a RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO: 1, and an mRNA including an ORF encoding a RSV F mutant A strain protein having the amino acid sequence of SEQ ID NO: 5. In one embodiment, the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 6. In another embodiment, the mRNA including the ORF encoding the RSV F mutant A strain protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the RSV F mutant A strain protein has the structure of 5' UTR-RSV F mutant A strain protein-3' UTR-poly(A) tail, wherein the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the poly(A) tail has a length of 50 nucleotides to 250 nucleotides. In one embodiment, the mRNA including the ORF encoding the 5' UTR-RSV F mutant A strain protein-3' UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7. In another embodiment, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant A strain protein has a nucleotide sequence having at least 80% identity to SEQ ID NO:7.

[0009] In one embodiment, the RSV vaccine composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

[0010] The present disclosure also provides a method for inducing an immune response against respiratory syncytial virus (RSV), comprising administering an effective amount of an RSV vaccine composition of the present disclosure to a subject in need thereof.

[0011] Additionally, the disclosure provides a respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccine composition, comprising a messenger ribonucleic acid (mRNA) including an open reading frame (ORF) encoding a RSV F mutant A strain protein having the amino acid sequence of SEQ ID NO: 5, an mRNA including an ORF encoding a RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO: 1, and an mRNA including an ORF encoding a hMPV F protein having the amino acid sequence of SEQ ID NO: 9. In one embodiment, the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 6, the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 2, and the ORF encoding the hMPV F protein has the nucleotide sequence of SEQ ID NO: 10. In another embodiment, the mRNA comprising an ORF encoding a RSV F mutant A strain protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the RSV F mutant A strain protein has the structure 5'UTR-RSV F mutant A strain protein-encoding ORF-3'UTR-poly(A) tail, wherein the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the mRNA comprising an ORF encoding a RSV F mutant B strain protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the RSV F mutant B strain protein has the structure 5'UTR-RSV F mutant B strain protein-encoding ORF-3'UTR-poly(A) tail, wherein the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 2. In another embodiment, the mRNA comprising the ORF encoding the hMPV F protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, so as to have a structure of 5' UTR-ORF encoding the hMPV F protein-3' UTR-poly(A) tail, and the ORF encoding the hMPV F protein has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the poly(A) tail has a length of 50 to 250 nucleotides.In some embodiments, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 3. In another embodiment, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-hMPV F protein has the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant A strain protein has a nucleotide sequence having at least 80% identity to SEQ ID NO: 7. In another embodiment, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein has a nucleotide sequence having at least 80% identity to SEQ ID NO: 3. In some embodiments, the mRNA having the structure of 5'UTR-hMPV F protein-encoding ORF-3'UTR-poly(A) tail has a nucleotide sequence that has at least 80% identity to SEQ ID NO:11.

[0012] In some embodiments, the RSV and hMPV vaccine compositions according to the present disclosure further comprise a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

[0013] The present disclosure also provides a method for inducing an immune response against respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), comprising administering an effective amount of the RSV and hMPV vaccine compositions of the present disclosure to a subject in need thereof. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the results of RSV-F IVT. [Figure 2] FIG. 1 shows the results of RSV-F Western blot. [Figure 3] FIG. 1 illustrates the immunogenicity and challenge study schedule of a combination RSV F mutant A strain mRNA vaccine and a RSV F mutant B strain mRNA vaccine in mice. [Figure 4] Figure 1 shows the results of RSV prefusion-specific IgG immunogenic responses. Ver 027 is a combination of RSV F mutant A strain mRNA vaccine and RSV F mutant B strain mRNA vaccine. [Figure 5] FIG. 3 shows titration of neutralizing antibodies (nAb) against RSV F mutant A strain (FIG. 3a) and RSV F mutant B strain (FIG. 3b). [Figure 6] 1 shows lung viral load after challenge with a combination of RSV F mutant A strain mRNA vaccine and RSV F mutant B strain mRNA vaccine. [Figure 7] FIG. 1 shows direct enumeration of F85-93 epitope-specific CD8+ T lymphocytes. [Figure 8] FIG. 1 shows the results of Western blotting using hMPV-F IVT and FLAG tag. DETAILED DESCRIPTION OF THE INVENTION

[0015] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, certain preferred methods, compositions, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described herein, as these may vary depending on routine experimentation and optimization. It should also be understood that the terminology used in the detailed description is for the purpose of describing particular forms or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0016] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, in case of conflict, the present specification, including definitions, will control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] As used herein, the term "comprise" and its linguistic variants indicate the presence of the recited feature(s), element(s), method step(s), etc., but do not exclude the presence of additional feature(s), element(s), method step(s), etc. In contrast, the term "consisting of" and its linguistic variants indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for impurities ordinarily associated therewith. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic characteristics of the composition, system, or method. Many embodiments herein are described using the open-ended term "comprising." Such embodiments encompass a number of the open-ended "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described in the specification using such terms.

[0019] As used herein, the term "respiratory syncytial virus (RSV) vaccine composition" refers to substances used to stimulate the production of antibodies and provide immunity against RSV.

[0020] As used herein, the term "respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccine composition" refers to substances used to stimulate the production of antibodies and provide immunity against RSV and hMPV.

[0021] As used herein, the term "messenger ribonucleic acid (mRNA)" refers to a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene and is read by ribosomes in the process of synthesizing proteins.

[0022] As used herein, the term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. In contrast, the term "modified" or "mutant" refers to a gene or gene product that exhibits altered characteristics compared to the wild-type gene or gene product. For example, a mutant DNA polymerase of the present invention is a DNA polymerase that exhibits reduced uracil detection activity.

[0023] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, and unless otherwise indicated, all D and L stereoisomers thereof, where the structure allows for such stereoisomeric forms.

[0024] Naturally occurring amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0025] Unnatural amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyproline ( "4Hyp"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines ("NAG") including N-methylglycine, N-methylisoleucine, N-alkylpentylglycines ("NAPG") including N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGly"), pipecolic acid, thioproline ("ThioP" or "tPro"), homolysine ("hLys"), and homoarginine ("hArg").

[0026] As used herein, the term "open reading frame (ORF)" refers to the nucleotide sequence between the start codon and the stop codon.

[0027] As used herein, the term "open reading frame (ORF) encoding" refers to a nucleotide coding sequence that encodes a polypeptide. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide.

[0028] As used herein, the term "T7 promoter" refers to the promoter derived from bacteriophage T7.

[0029] As used herein, the term "5' untranslated region (UTR)" refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (the first codon of an mRNA transcript that is translated by ribosomes) that does not encode a polypeptide.

[0030] As used herein, the term "3' untranslated region (UTR)" refers to the region of an mRNA immediately downstream (i.e., 3') from the stop codon (i.e., the codon of the mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0031] As used herein, the term "poly(A) tail" refers to the "tail" of an mRNA, i.e., the long stretch of adenine nucleotides added to the 3' end.

[0032] As used herein, the term "pharmaceutically acceptable carrier" refers to any substance or vehicle suitable for delivering an mRNA vaccine to a suitable in vivo or ex vivo site. Such carriers may include, but are not limited to, adjuvants, excipients, lipid particles, etc.

[0033] As used herein, the term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer scale (e.g., 1 nm to 1000 nm). In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver mRNA vaccines to desired target sites (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the mRNA vaccine can be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cellular machinery, such as a harmful immune response. In some embodiments, the lipid nanoparticles have an average diameter of 50 nm to 200 nm. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of approximately 20% to 60% cationic lipid, 0.5% to 15% PEG-modified lipid, 25% to 55% sterol, and 25% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0034] As used herein, the term "inducing an immune response against respiratory syncytial virus (RSV)" refers to providing protective immunity and / or vaccinating a subject against RSV infection for prophylactic purposes, and to generating a desired immune response or effect against RSV infection in a subject in need thereof for therapeutic purposes. As used herein, the term "protective immunity" or "protective immune response" means that a vaccinated subject is able to control infection by the pathogen against which the vaccine was administered. Typically, subjects who develop a "protective immune response" develop only mild to moderate clinical symptoms, or no symptoms at all.

[0035] An "effective amount" of an RSV vaccine composition (e.g., mRNA) or RSV and hMPV vaccine composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size and degree of nucleoside modification) and other components of the vaccine, as well as other determinants. Generally, an effective amount of an RSV vaccine (e.g., mRNA) or RSV and hMPV vaccine composition induces or boosts an immune response in response to intracellular antigen production, preferably more efficiently than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production can be demonstrated by increased cell transfection (percentage of cells transfected with RNA, e.g., mRNA, vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., as indicated by an increased duration of protein translation from the modified polynucleotide), or a change in the antigen-specific immune response of the host cell.

[0036] As used herein, the term "X% identity to SEQ ID NO: Y" or "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomer subunits. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only by conservative and / or semi-conservative amino acid substitutions. "Percent sequence identity" (or "percent sequence similarity") is calculated by (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid occurs in both sequences, a similar amino acid occurs in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptide A and peptide B are both 20 amino acids long and have identical amino acids at all positions except one, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical properties (e.g., both are acidic), peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to those in a portion of peptide C, peptides C and D will have 70% sequence identity, but peptide D will have 93.3% sequence identity over the optimal comparison window of peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0037] As used herein, the term "a nucleotide sequence having at least X% identity to sequence number Y and encoding a Z protein" means that the nucleotide sequence satisfies two distinct requirements: having at least X% identity to sequence number Y and encoding a Z protein.

[0038] As used herein, the terms "about," "approximate," "at or about," and "substantially" mean that the relevant amount or value may be the exact value set forth in the claims or taught herein, or a value that will produce an equivalent result or effect. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, if desired, to produce an equivalent result or effect, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those skilled in the art. In some circumstances, it may not be possible to reasonably determine a value that will produce an equivalent result or effect. In such cases, generally, as used herein, "about" and "at or about" are understood to mean a ±10% variation from the nominal value indicated, unless otherwise indicated or implied. In general, amounts, sizes, formulations, parameters, or other quantities or characteristics are "about," "approximate," or "at or about," whether or not expressly stated as such. When "about," "approximate," or "at or about" is used before or after a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless otherwise specifically stated.

[0039] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal, any mammalian subject, or cells thereof, suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0040] Detailed Description 1. Respiratory syncytial virus (RSV) vaccine composition, or RSV and human metapneumovirus (hMPV) vaccine composition The present disclosure provides a respiratory syncytial virus (RSV) vaccine composition comprising a messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding a RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO:1.

[0041] In the RSV vaccine composition, the RSV F mutant B strain protein may have an amino acid sequence having at least 80% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 85% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 95% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 96% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 97% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 98% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 99% identity to SEQ ID NO: 1.

[0042] In the RSV vaccine composition, the mRNA containing the ORF encoding the RSV F mutant B strain protein may further comprise a 5' untranslated region (UTR), a 3' UTR, and a poly (A) tail, such that the ORF encoding the RSV F mutant B strain protein has the structure 5' UTR-RSV F mutant B strain protein encoding ORF-3' UTR-poly (A) tail, wherein the ORF may have the nucleotide sequence of SEQ ID NO: 2 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 2).

[0043] In the RSV vaccine composition, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein may have the nucleotide sequence of SEQ ID NO: 3 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 3).

[0044] The present disclosure also provides a RSV vaccine composition comprising an mRNA containing an ORF encoding a RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO: 1 and an mRNA containing an ORF encoding a RSV F mutant A strain protein having the amino acid sequence of SEQ ID NO: 5. The RSV vaccine composition described above, which contains both an mRNA encoding an ORF of the RSV F mutant B strain protein and an mRNA encoding an ORF of the RSV F mutant A strain protein, provides greater protection against the RSV F mutant B strain than an RSV vaccine composition containing only an mRNA encoding an ORF of the RSV F mutant A strain protein. The latter provides protection against the RSV F mutant B strain based solely on cross-reactivity.

[0045] In the RSV vaccine composition, the RSV F mutant A strain protein may have an amino acid sequence having at least 80% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 85% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 90% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 95% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 96% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 97% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 98% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 99% identity to SEQ ID NO:5.

[0046] In the RSV vaccine composition, the mRNA containing the ORF encoding the RSV F mutant A strain protein may further comprise a 5' untranslated region (UTR), a 3' UTR, and a poly (A) tail, such that the ORF encoding the RSV F mutant A strain protein has the structure 5' UTR-RSV F mutant A strain protein encoding ORF-3' UTR-poly (A) tail, wherein the ORF may have the nucleotide sequence of SEQ ID NO: 6 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 6).

[0047] In the RSV vaccine composition, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant A strain protein may have the nucleotide sequence of SEQ ID NO: 7 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 7).

[0048] The present disclosure also provides a respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccine composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an RSV F mutant A strain protein having the amino acid sequence of SEQ ID NO:5, an mRNA comprising an ORF encoding an RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO:1, and an mRNA comprising an ORF encoding an hMPV F protein having the amino acid sequence of SEQ ID NO:9.

[0049] In the RSV and hMPV vaccine compositions, the RSV F mutant A strain protein may have an amino acid sequence having at least 80% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 85% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 90% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 95% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 96% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 97% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 98% identity to SEQ ID NO:5. In another embodiment, the RSV F mutant A strain protein has an amino acid sequence having at least 99% identity to SEQ ID NO:5.

[0050] In the RSV and hMPV vaccine compositions, the RSV F mutant B strain protein may have an amino acid sequence having at least 80% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 85% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 95% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 96% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 97% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 98% identity to SEQ ID NO: 1. In another embodiment, the RSV F mutant B strain protein has an amino acid sequence having at least 99% identity to SEQ ID NO: 1.

[0051] In the RSV and hMPV vaccine compositions, the hMPV F protein may have an amino acid sequence at least 80% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 85% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 90% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 95% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 96% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 97% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 98% identical to SEQ ID NO:9. In another embodiment, the hMPV F protein has an amino acid sequence at least 99% identical to SEQ ID NO:9.

[0052] In one embodiment, the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 6, the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 2, and the ORF encoding the hMPV F protein has the nucleotide sequence of SEQ ID NO: 10. In another embodiment, the mRNA containing the ORF encoding the RSV F mutant A strain protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the RSV F mutant A strain protein has the structure 5' UTR-RSV F mutant A strain protein encoding ORF-3' UTR-poly(A) tail, wherein the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the mRNA comprising an ORF encoding a RSV F mutant B strain protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the RSV F mutant B strain protein has the structure 5'UTR-RSV F mutant B strain protein-encoding ORF-3'UTR-poly(A) tail, wherein the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 2. In another embodiment, the mRNA comprising an ORF encoding the hMPV F protein further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF encoding the hMPV F protein has the structure 5'UTR-hMPV F protein-encoding ORF-3'UTR-poly(A) tail, wherein the ORF encoding the hMPV F protein has the nucleotide sequence of SEQ ID NO: 10.

[0053] In the RSV and hMPV vaccine compositions, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 80% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 85% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 90% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 95% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 96% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 97% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 98% identity to SEQ ID NO:6. In another embodiment, the ORF encoding the RSV F mutant A strain protein may have a nucleotide sequence having at least 99% identity to SEQ ID NO:6.

[0054] In the RSV and hMPV vaccine compositions, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 80% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 85% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 90% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 95% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 96% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 97% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 98% identity to SEQ ID NO:2. In another embodiment, the ORF encoding the RSV F mutant B strain protein may have a nucleotide sequence having at least 99% identity to SEQ ID NO:2.

[0055] In the RSV and hMPV vaccine compositions, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 80% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 85% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 90% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 95% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 96% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 97% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein may have a nucleotide sequence that is at least 98% identical to SEQ ID NO: 10. In another embodiment, the ORF encoding the hMPV F protein can have a nucleotide sequence having at least 99% identity to SEQ ID NO:10.

[0056] In one embodiment, the poly(A) tail has a length of 50 nucleotides to 250 nucleotides. In some embodiments, an mRNA having the structure ORF-3'UTR-poly(A) tail encoding a 5'UTR-RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO: 7. In some embodiments, an mRNA having the structure ORF-3'UTR-poly(A) tail encoding a 5'UTR-RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO: 3. In another embodiment, an mRNA having the structure ORF-3'UTR-poly(A) tail encoding a 5'UTR-hMPV F protein has the nucleotide sequence of SEQ ID NO: 11. In some embodiments, an mRNA having the structure ORF-3'UTR-poly(A) tail encoding a 5'UTR-RSV F mutant A strain protein has a nucleotide sequence having at least 80% identity to SEQ ID NO: 7 (or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 7). In another embodiment, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein has a nucleotide sequence having at least 80% identity to SEQ ID NO: 3 (or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 3). In some embodiments, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-hMPV F protein has a nucleotide sequence having at least 80% identity to SEQ ID NO: 11 (or a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 11).

[0057] In one embodiment, the poly(A) tail has a length of 50 to 250 nucleotides. In another embodiment, the poly(A) tail has a length of 100 to 200 nucleotides. In another embodiment, the poly(A) tail has a length of 110 to 150 nucleotides. In another embodiment, the poly(A) tail has a length of 115 to 125 nucleotides. In another embodiment, the poly(A) tail has a length of 116 to 124 nucleotides. In another embodiment, the poly(A) tail has a length of 117 to 123 nucleotides. In another embodiment, the poly(A) tail has a length of 118 to 122 nucleotides. In another embodiment, the poly(A) tail has a length of 119 to 122 nucleotides. In another embodiment, the poly(A) tail has a length of 115 nucleotides. In another embodiment, the poly(A) tail has a length of 116 nucleotides. In another embodiment, the poly(A) tail has a length of 117 nucleotides. In another embodiment, the poly(A) tail has a length of 118 nucleotides. In another embodiment, the poly(A) tail has a length of 119 nucleotides. In another embodiment, the poly(A) tail has a length of 120 nucleotides. In another embodiment, the poly(A) tail has a length of 121 nucleotides. In another embodiment, the poly(A) tail has a length of 122 nucleotides. In another embodiment, the poly(A) tail has a length of 123 nucleotides. In another embodiment, the poly(A) tail has a length of 124 nucleotides. In another embodiment, the poly(A) tail has a length of 125 nucleotides.

[0058] In one embodiment, the mRNA of the present disclosure may contain at least one chemical modification selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In another embodiment, the chemical modification is at the 5-position of uracil. In another embodiment, the chemical modification is N1-methylpseudouridine. In another embodiment, the chemical modification is N1-ethylpseudouridine.

[0059] In one embodiment, the RSV vaccine composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutically acceptable carrier may comprise any substance or vehicle suitable for delivering the mRNA vaccine to a suitable in vivo or ex vivo site. Such carriers may include, but are not limited to, adjuvants, excipients, lipid particles, etc. The lipid nanoparticles may be particles having at least one dimension on the nanometer scale (e.g., 1 nm to 1000 nm). In some embodiments, the lipid nanoparticles are included in a formulation that can be used to deliver the mRNA vaccine to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the mRNA vaccine can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cell's machinery, such as a harmful immune response. In some embodiments, the lipid nanoparticles have an average diameter of 50 nm to 200 nm. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of about 20%-60% cationic lipid, 0.5%-15% PEG-modified lipid, 25%-55% sterol, and 25% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0060] In one embodiment, the lipid nanoparticles comprise (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG lipid, such as PEG-DMG or PEG-cDMA, in a molar ratio of about 20%-60% cationic lipid:5%-25% neutral lipid:25%-55% sterol:0.5%-15% PEG lipid.

[0061] In one embodiment, the lipid nanoparticles comprise about 25% to about 75% on a molar basis, e.g., about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50%, or about 40% on a molar basis, of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0062] In one embodiment, the lipid nanoparticles contain about 0.5% to about 15% neutral lipid on a molar basis, e.g., about 3% to about 12%, about 5% to about 10%, or about 15%, about 10%, or about 7.5%. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation contains about 5% to about 50% sterol on a molar basis (e.g., about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31%). An exemplary sterol is cholesterol. In some embodiments, the formulation comprises about 0.5% to about 20% PEG-lipid or PEG-modified lipid on a molar basis (e.g., about 0.5% to about 10%, about 0.5% to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis). In some embodiments, the PEG-lipid or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2000 Da. In other embodiments, the PEG-lipid or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2000, e.g., around 1500 Da, around 1000 Da, or around 500 Da. Examples of PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG) and PEG-cDMA.

[0063] In one embodiment, the lipid nanoparticles comprise, on a molar basis, 25% to 75% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5% to 15% of a neutral lipid, 5% to 50% of a sterol, and 0.5% to 20% of a PEG lipid or a PEG-modified lipid.

[0064] In one embodiment, the lipid nanoparticles comprise, on a molar basis, 35% to 65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3% to 12% of a neutral lipid, 15% to 45% of a sterol, and 0.5% to 10% of a PEG lipid or a PEG-modified lipid.

[0065] In one embodiment, the lipid nanoparticles comprise, on a molar basis, 45% to 65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5% to 10% of a neutral lipid, 25% to 40% of a sterol, and 0.5% to 10% of a PEG lipid or a PEG-modified lipid.

[0066] In one embodiment, the lipid nanoparticles comprise about 60% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% neutral lipid, about 31% sterol, and about 1.5% PEG lipid or PEG-modified lipid.

[0067] In one embodiment, the lipid nanoparticles comprise about 50% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 38.5% sterol, and about 1.5% PEG lipid or PEG-modified lipid.

[0068] In one embodiment, the lipid nanoparticles comprise about 50% by molar cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 35% sterol, about 4.5% or about 5% PEG lipid or PEG-modified lipid, and about 0.5% targeting lipid.

[0069] In one embodiment, the lipid nanoparticles comprise about 40% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% neutral lipid, about 40% sterol, and about 5% PEG lipid or PEG-modified lipid.

[0070] In one embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure can be delivered, localized, and / or concentrated to a specific location using the delivery methods described below. As a non-limiting example, empty polymer particles can be administered to a subject before, simultaneously with, or after the RSV vaccine composition of the present disclosure is delivered to the subject. Upon contact with the subject, the empty polymer particles undergo a volume change and remain, become embedded, immobilized, or become trapped at a specific location within the subject.

[0071] In another embodiment, the RSV vaccine composition of the present disclosure can be formulated into an active substance release system. For example, the active substance release system can include at least one nanoparticle bound to an oligonucleotide inhibitor chain hybridized with a catalytically active nucleic acid and a compound bound to at least one substrate molecule bound to a therapeutically active substance (e.g., a polynucleotide described herein), wherein the therapeutically active substance is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0072] In another embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure can be formulated into nanoparticles comprising an inner core comprising non-cellular material and an outer surface comprising a cellular membrane, which may be derived from a cell or a virus.

[0073] In another embodiment, the RSV vaccine compositions or RSV and hMPV vaccine compositions of the present disclosure can be formulated into porous nanoparticle-supported lipid bilayers (protocells).

[0074] In another embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure can be formulated into polymeric nanoparticles with high glass transition temperatures.

[0075] In another embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure can be formulated into nanoparticles for use in imaging. As a non-limiting example, a liposome can comprise gadolinium(III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-acetate and a neutral, fully saturated phospholipid component.

[0076] The nanoparticles of the present disclosure may further comprise nutrients, such as, but not limited to, nutrients whose deficiency can cause adverse health effects ranging from anemia to neural tube defects. By way of non-limiting example, the nutrient can be iron in the form of ferrous salts, ferric salts, or elemental iron, iodine, folic acid, vitamins, or micronutrients.

[0077] In another embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure can be formulated into swellable nanoparticles.

[0078] In another embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure can be formulated in polyanhydride nanoparticles.

[0079] The nanoparticles and microparticles of the present disclosure can be geometrically engineered to modulate macrophage and / or immune responses. In some embodiments, the geometrically engineered particles can have various shapes, sizes, and / or surface charges to incorporate polynucleotides of the present disclosure for targeted delivery, such as, but not limited to, pulmonary delivery. Other physical features that the geometrically engineered particles can have include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, and charge, which can alter interactions with cells and tissues.

[0080] In another embodiment, the nanoparticles of the present disclosure may be water-soluble nanoparticles. The nanoparticles may be inorganic nanoparticles that are compact and have zwitterionic ligands to exhibit good water solubility. The nanoparticles may also have a small hydrodynamic diameter (HD), stability over time, pH, and salt concentration, and low levels of nonspecific protein binding.

[0081] In some embodiments, the nanoparticles of the present disclosure are stealth nanoparticles or target-specific stealth nanoparticles. In some embodiments, the stealth nanoparticles or target-specific stealth nanoparticles can comprise a polymer matrix. The polymer matrix can comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or a combination thereof.

[0082] In one embodiment, the nanoparticles of the present disclosure may be nanoparticle-nucleic acid hybrid structures having a high-density nucleic acid layer. The nanoparticles of the present disclosure may comprise nucleic acids, such as, but not limited to, polynucleotides described herein and / or known in the art.

[0083] In one embodiment, at least one of the nanoparticles of the present disclosure may be embedded in the core of the nanostructure or coated with a low-density porous 3D structure or coating capable of carrying or associating at least one payload within or on the surface of the nanostructure.

[0084] In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA of the present disclosure.

[0085] 2. A method for inducing an immune response against respiratory syncytial virus (RSV), or a method for inducing an immune response against RSV and human metapneumovirus (hMPV) The present disclosure also provides a method for inducing an immune response against respiratory syncytial virus (RSV), comprising administering an effective amount of an RSV vaccine composition of the present disclosure to a subject in need thereof.

[0086] The present disclosure also provides methods for inducing an immune response against RSV and human metapneumovirus (hMPV), comprising administering to a subject in need thereof an effective amount of the RSV and hMPV vaccine compositions of the present disclosure.

[0087] In one embodiment, an effective amount of the RSV vaccine composition or RSV and hMPV vaccine composition (e.g., mRNA) is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size and degree of modified nucleosides) and other components of the vaccine, as well as other determinants. Generally, an effective amount of the RSV vaccine composition or RSV and hMPV vaccine composition (e.g., mRNA) induces or boosts an immune response in response to intracellular antigen production, preferably more efficiently than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production can be demonstrated by increased cell transfection (percentage of cells transfected with RNA, e.g., mRNA, vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., indicated by an increased duration of protein translation from the modified polynucleotide), or a change in the antigen-specific immune response of the host cell.

[0088] An effective amount (immunogenically effective amount) of the RSV vaccine composition or RSV and hMPV vaccine composition is typically administered intramuscularly or subcutaneously. Therefore, the RSV vaccine composition is typically formulated for intramuscular or subcutaneous injection, and for purposes of the present invention, it is formulated without an adjuvant, preferably without any adjuvant. However, other modes of administration, such as intravenous, cutaneous, intradermal, or intranasal, can also be envisioned. For intravenous, cutaneous, or subcutaneous injection, the adenoviral vector is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Similarly, the isolated envelope polypeptide is in the form of a parenterally acceptable solution that has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.

[0089] In certain embodiments, an effective amount (immunogenically effective amount) of the RSV vaccine composition or RSV and hMPV vaccine composition is administered intramuscularly. Intramuscular administration can be achieved by injecting a suspension of the adenoviral vector and / or envelope polypeptide using a needle. Alternatively, a needleless injection device is used to administer the composition (e.g., using Biojector™) or a lyophilized powder containing the vaccine.

[0090] In one embodiment, the priming immunization and / or the boosting administration, preferably both the priming administration and the boosting administration, further comprises administering one or more adenoviral vectors encoding one or more additional RSV antigens or RSV and hMPV antigens.

[0091] The timing of priming and boosting immunizations is not particularly limited. For example, the vaccine composition can be administered for priming immunization and then re-administered before administering the vaccine composition for boosting immunization. Further administration of the vaccine composition for further boosting immunization is also contemplated. In certain embodiments, the booster vaccine is first administered about 1 to 12 weeks after the initial administration of the primer vaccine, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks later. In another embodiment, the booster vaccine is first administered about 12 to 52 weeks after the initial administration of the primer vaccine, for example, about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 weeks later. Those skilled in the art can modify the exact timing, frequency of administration, dosage, etc. of the priming and boosting vaccines based on the teachings herein and general knowledge in the art.

[0092] In one embodiment, the RSV vaccine composition or RSV and hMPV vaccine composition can include a first mRNA and a second mRNA described herein formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC, and PEG2000-DMG, trisodium citrate buffer, sucrose, and water for injection. As a non-limiting example, the composition can include 2.0 mg / mL of drug substance, 21.8 mg / mL of MC3, 10.1 mg / mL of cholesterol, 5.4 mg / mL of DSPC, 2.7 mg / mL of PEG2000-DMG, 5.16 mg / mL of trisodium citrate, 71 mg / mL of sucrose, and 1.0 mL of water for injection.

[0093] In one embodiment, a method for inducing an immune response to RSV comprises administering an effective amount of the RSV vaccine composition of the present disclosure to a subject in need thereof. In the RSV vaccine composition, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein may have the nucleotide sequence of SEQ ID NO: 3 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 3).

[0094] In another embodiment, the mRNA having the structure of ORF-3'UTR-poly (A) tail encoding the 5'UTR-RSV F mutant B strain protein included in the RSV vaccine composition may have the nucleotide sequence of SEQ ID NO: 3 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 3), and the mRNA having the structure of ORF-3'UTR-poly (A) tail encoding the 5'UTR-RSV F mutant A strain protein may have the nucleotide sequence of SEQ ID NO: 7 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 7).

[0095] In another embodiment, a method for inducing an immune response to RSV and hMPV comprises administering an effective amount of the RSV and hMPV vaccine composition of the present disclosure to a subject in need thereof. In the RSV and hMPV vaccine composition, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant A strain protein can have the nucleotide sequence of SEQ ID NO: 7 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 7). Additionally, the mRNA having the structure ORF-3'UTR-poly(A) tail encoding the 5'UTR-RSV F mutant B strain protein can have the nucleotide sequence of SEQ ID NO: 3 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 3). Furthermore, an mRNA having the structure of ORF-3'UTR-poly(A) tail encoding a 5'UTR-hMPV protein can have the nucleotide sequence of SEQ ID NO: 11 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 11).

[0096] 3. Sequence information 1) Protein sequence of the ORF encoded by RSV F mutant B strain mRNA SEQ ID NO: 1 MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0097] 2) RSV F mutant B strain mRNA sequence (ORF) SEQ ID NO: 2

[0098] 3) RSV F mutant B strain mRNA sequence (5'-UTR-ORF-3'UTR-poly(A) tail) SEQ ID NO: 3

[0099] 4) Sequence of pUC57 plasmid encoding RSV F mutant B strain mRNA sequence (mRNA sequence underlined) SEQ ID NO:4 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTTGAAATACCGCACAGATGCGTAAGGAGAAAATAC CGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGATTCTAGATTAATACGACTCACTATA AGGCCGGCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGGAACTGCTGATCCACAGATCCAGCGCTATTTTCCTGACACTGGCCATCAATGCCCTGTACCTGACAAGCAGCCAGAACATCACAGAGGAATTCTACCAGAGTACCTGTAGCGCTGTGTCTCGGGGATACCTGAGCGCCCTGCGGACCGGTTGGTACACCAGCGTGATCACAATCGAGCTGAGTAACATCAAGGAGACAAAATGCAATGGCACTGACACCAAGGTGAAACTTATCAAACAGGAGCTGGATAAGTACAAGAATGCCGTGACAGAACTGCAGCTGCTGATGCAGAATACCCCCGCCGTGAACAACAGAGCCAGACGGGAAGCCCCTCAATACATGAACTACACAATCAACACCACAAAGAACCTGAACGTGTCTATCTCCAAGAAGCGCAAGCGGAGATTCCTGGGCTTCCTGTTGGGAGTGGGCTCTGCCATCGCCAGCGGCATCGCAGTGTGCAAGGTGCTGCACCTGGAAGGAGAAGTGAACAAGATCAAGAATGCCCTGCAGCTGACCAACAAGGCCGTGGTCTCTCTGAGCAATGGCGTGAGCGTGCTGACCTTTAGAGTGCTGGACCTGAAGAACTACATCAACAACCAGCTGCTGCCTATGCTGAACAGACAGTCTTGTAGAATCAGCAACATCGAAACCGTGATCGAGTTCCAGCAGAAGAACTCCAGGCTGCTGGAAATCACAAGAGAGTTTAGCGTGAACGCCGGCGTCACCACCCCCCTGAGCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATTAACGACATGCCTATCACCAATGACCAGAAAAAGCTAATGAGCAGCAATGTGCAAATCGTGCGGCAGCAGTCCTACAGCATCATGTGCATCATCAAGGAAGAGGTCCTGGCCTACGTGGTACAACTGCCTATCTACGGCGTGATCGACACCCCTTGTTGGAAGCTCCATACCAGCCCTCTGTGCACAACAAACATCAAAGAAGGCAGCAATATCTGCCTGACAAGAACAGATAGAGGCTGGTATTGTGACAACGCTGGCTCCGTCTCCTTCTTCCCTCAGGCCGACACCTGCAAGGTGCAGAGCAACCGCGTGTTCTGCGATACCATGAACAGCTTAACACTGCCCAGCGAGGTGTCTCTCTGTAACACCGACATCTTCAACTCTAAATACGACTGCAAGATCATGACCTCTAAGACTGATATCAGCAGCAGCGTGATTACCTCCCTGGGCGCCATCGTGTCTTGCTACGGCAAGACAAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCGTGGACACCGTTAGCGTGGGCAACACACTGTATTACGTAAATAAACTTGAGGGCAAGAATCTGTACGTGAAGGGCGAGCCTATCATCAACTACTACGACCCACTGGTGTTTCCATCTGATGAGTTCGACGCCTCCATCTCCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCTTTTATCAGAAGAAGCGATGAGCTGCTGCACAACGTGAATACCGGAAAAAGCACCACCAACATCATGATCACCGCCATCATTATCGTTATCATCGTGGTGCTGCTGAGTCTGATCGCCATTGGACTGCTGCTGTATTGCAAAGCCAAGAACACCCCCGTGACCCTGAGCAAGGACCAGCTGAGCGGCATCAACAACATAGCTTTCTCCAAGTGACTCGAGTAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0100] 5) Protein sequence of the ORF encoded by RSV F mutant A strain mRNA SEQ ID NO:5 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLG VGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYS IMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0101] 6) RSV F mutant A strain mRNA sequence (ORF) SEQ ID NO:6

[0102] 7) RSV F mutant A strain mRNA sequence (5'-UTR-ORF-3'UTR-poly(A) tail) SEQ ID NO:7

[0103] 8) Sequence of pUC57 plasmid encoding RSV F mutant A strain mRNA sequence (RSV F mutant A strain mRNA sequence is underlined) SEQ ID NO:8 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGAAATACCGCACAGATGCGTAAGGAGAAAA TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCGGCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGGAACTGCTGATCCTGAAGGCCAACGCTATCACAACCATCCTCACCGCCGTGACATTCTGCTTCGCCAGCGGCCAGAACATCACCGAAGAGTTCTACCAGAGCACCTGCTCCGCTGTGTCTAAAGGGTACCTGTCCGCCCTGAGAACCGGCTGGTATACCAGCGTGATTACGATTGAGCTGAGCAACATCAAGAAGAACAAGTGCAACGGAACAGACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGATAAGTATAAGAACGCCGTGACCGAGTTGCAGCTGCTCATGCAGTCTACACAGGCCACAAACAATCGGGCCAGAAGAGAGCTGCCTAGATTCATGAACTACACCCTGAACAACGCCAAGAAGACAAATGTGACCCTGAGCAAGAAAAGAAAGCGGAGATTCCTGGGCTTCCTGCTCGGCGTTGGCTCTGCCATCGCCAGCGGAGTCGCCGTGTGCAAAGTGCTGCACCTGGAAGGAGAAGTGAACAAGATCAAGAGCGCCCTGCTGTCTACGAACAAAGCCGTGGTGAGCCTGTCCAACGGCGTGAGCGTGCTGACCTTCAAGGTGCTGGACCTGAAGAACTACATCGATAAGCAACTGCTGCCAATCCTGAACAAGCAGTCTTGTAGCATCAGCAATATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACACGGGAATTTAGCGTCAACGCCGGTGTGACCACCCCTGTGTCTACCTACATGCTGACCAATAGCGAGCTGCTGAGCCTGATCAATGACATGCCTATTACCAACGACCAAAAGAAACTGATGAGCAACAATGTACAAATCGTTAGACAGCAGAGCTACTCCATCATGTGCATCATCAAGGAGGAAGTGCTGGCCTACGTCGTGCAACTGCCTCTGTACGGCGTGATCGACACCCCTTGTTGGAAGCTGCACACGAGCCCTCTGTGCACAACTAATACAAAGGAAGGCAGCAACATCTGCCTGACCAGAACAGATAGAGGCTGGTACTGCGACAACGCTGGATCTGTCAGCTTTTTCCCTCAGGCTGAAACCTGTAAAGTGCAGTCCAACCGCGTGTTTTGCGATACAATGAACTCTCTGACACTGCCTAGCGAGGTGAACCTGTGTAATGTGGACATCTTCAACCCCAAGTACGATTGCAAGATCATGACAAGCAAGACCGATGTGTCTAGCAGCGTGATTACCAGCCTGGGCGCCATCGTGTCATGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAAAATCGGGGCATCATCAAAACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAAGGAGTGGACACCGTCTCCGTGGGCAACACCCTGTACTACGTGAACAAGCAGGAGGGCAAGTCTCTGTACGTGAAGGGCGAGCCAATCATTAACTTCTACGACCCCCTGGTGTTCCCCTCTGATGAGTTCGACGCCTCCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAACGTCAATGCCGGCAAAAGCACCACCAACATCATGATCACCACCATCATCATCGTGATCATTGTGATCCTGCTGAGTCTGATCGCTGTGGGCCTGCTGCTGTATTGTAAAGCTAGGTCCACCCCCGTGACACTTAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCATTTAGCAACTGATAAAGCTGGAGCCTCGGTGGCCTTGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0104] 9) Protein sequence of the ORF encoded by hMPV-F mRNA SEQ ID NO:9 MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEV TAIKNALKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIY MVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLN KGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIVIILIAVLGSSMILVSICIIIKKTKKPTGAPPELSGVTNNGFIPHS

[0105] 10) hMPV-F mRNA (ORF) SEQ ID NO: 10

[0106] 11) hMPV-F mRNA sequence (5'-UTR-ORF-3'UTR-poly(A) tail) SEQ ID NO: 11

[0107] 12) Sequence of pUC57 plasmid encoding hMPV-F mRNA (hMPV-F mRNA sequence is underlined) SEQ ID NO: 12 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGAAATACCGCACAGATGCGTAAGGAGAAAA TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCGGCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGAGCTGGAAGGTGGTGATCATCTTCAGCCTGCTGATCACCCCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAAAGCTGCTCTACCATCACAGAGGGCTACCTGAGTGTGCTGCGGACCGGCTGGTACACAAACGTGTTTACCCTGGAAGTGGGCGACGTGGAAAACCTGACATGCGCCGATGGCCCCAGCCTGATTAAGACAGAGCTGGACCTGACAAAGAGCGCCCTGAGAGAACTGAAGACAGTCAGCGCTGATCAACTGGCCAGGGAGGAGCAGATCGAGAACCCCAGACAGTCCAGATTCGTGCTCGGTGCCATCGCCCTGGGCGTGGCGACAGCTGCCGCTGTTACCGCTGGCGTGGCTATCGCCAAGACCATCAGACTGGAATCTGAGGTGACCGCCATCAAGAATGCCCTGAAGAAGACCAATGAGGCCGTGTCTACACTGGGAAATGGCGTGCGGGTGCTGGCTACAGCCGTCCGGGAACTGAAAGACTTCGTGTCCAAGAACTTGACCAGAGCCATCAACAAGAACAAGTGCGATATCGACGACCTGAAGATGGCCGTGAGCTTCAGCCAGTTCAACCGGCGGTTCCTGAACGTTGTGCGGCAGTTTAGCGACAACGCCGGAATCACCCCAGCCATCAGCCTGGACCTGATGACCGACGCCGAGCTGGCCAGAGCCGTGAGCAACATGCCTACAAGCGCCGGCCAGATCAAGCTGATGCTGGAGAATCGGGCCATGGTGAGAAGAAAGGGCTTTGGCATCCTGATCGGCGTGTACGGCAGCAGCGTGATCTACATGGTGCAGCTGCCTATCTTCGGCGTGATCGATACCCCTTGCTGGATCGTGAAGGCCGCCCCTAGCTGTAGCGAGAAAAAGGGAAACTACGCCTGTCTGCTGAGAGAAGATCAGGGCTGGTATTGCCAGAACGCCGGCAGCACCGTGTACTACCCCAACGAGAAGGACTGCGAGACAAGAGGCGATCACGTGTTCTGCGACACCGCCGCCGGCATCAACGTTGCTGAGCAAAGCAAGGAATGCAACATCAACATCAGCACCACCAACTACCCTTGTAAAGTGAGCACCGGAAGACACCCCATCTCCATGGTCGCACTCTCCCCTCTGGGCGCCCTGGTCGCCTGCTACAAGGGCGTGTCTTGTAGCATCGGAAGCAACCGCGTTGGCATCATCAAGCAGCTGAACAAAGGATGTTCTTATATCACCAACCAGGATGCCGACACCGTGACAATCGACAATACCGTCTACCAGCTGAGCAAGGTGGAAGGCGAGCAGCACGTGATTAAGGGCAGACCTGTGTCCTCTAGCTTCGACCCCGTGAAGTTCCCCGAGGACCAGTTCAATGTGGCCCTGGATCAAGTGTTTGAGAACATCGAGAACAGCCAGGCCCTGGTGGACCAGAGCAATAGAATCCTGTCCTCCGCTGAGAAAGGCAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCTCTAGCATGATCCTGGTGTCTATCTGCATTATTATCAAGAAAACCAAGAAGCCTACCGGCGCTCCACCTGAGCTGAGCGGAGTGACCAACAACGGCTTCATCCCTCATTCTTGACTCGAGTAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [Example]

[0108] Example 1 - DNA template used for in vitro transcription and protein expression of the RSV F gene The DNA construct used for RSV F mutant B strain mRNA production (in vitro transcription, IVT) consisted of 1) a T7 promoter, 2) a 5' untranslated region (UTR), 3) an open reading frame (ORF) of a modified human RSV B strain membrane glycoprotein DNA (GenBank: MZ515553.1) (RSV F mutant B strain), 4) a 3' UTR, and 5) 120 bases of polyadenine (polyA). The sequences of the 5' UTR and 3' UTR were from human hemoglobin subunit alpha 1 (HBA1) mRNA (GenBank: NM_000558.5). The sequence of the pUC57 plasmid encoding the RSV F mutant B strain mRNA is shown in SEQ ID NO: 4.

[0109] The DNA fragment was synthesized by GenScript (Piscataway, NJ: https: / / www.genscript.com / vector / SD1176-pUC57_plasmid_DNA.html) and subcloned into the pUC57-Kan vector. For the stabilized prefusion form of the RSV-F protein, a mutant RSV-F (Fmut) with four amino acid substitutions (S155C, S190F, V207L, and S290C) was generated by mutagenesis of the RSV-F clone by GenScript (Fig. 1, top).

[0110] The DNA construct used for RSV F mutant A strain mRNA production (in vitro transcription, IVT) consisted of 1) a T7 promoter, 2) a 5' untranslated region (UTR), 3) an open reading frame (ORF) of the RSV F mutant A strain modified from human RSV A2 strain membrane glycoprotein DNA (GenBank: KT992094.1), 4) a 3'UTR, and 5) 120 bases of polyadenine (polyA). The sequences of the 5'UTR and 3'UTR were derived from human hemoglobin subunit alpha 1 (HBA1) mRNA (GenBank: NM_000558.5). The sequence of the pUC57 plasmid encoding the RSV F mutant A strain is shown in SEQ ID NO:8.

[0111] The DNA fragment was synthesized by GenScript (Piscataway, NJ: https: / / www.genscript.com / vector / SD1176-pUC57_plasmid_DNA.html) and subcloned into the pUC57-Kan vector. For the stabilized prefusion form of the RSV-F protein, a mutant RSV-F (Fmut) with four amino acid substitutions (S155C, S190F, V207L, and S290C) was generated by mutagenesis of the RSV-F clone by GenScript.

[0112] Example 2 - In vitro transcription (IVT) For RSV F IVT, the plasmid vector was linearized with the restriction enzyme PmeI or SapI (New England Biolabs). N1-methylpseudouridine (m1Ψ) was purchased from BOC Sciences (Shirley, NY). IVT conditions followed the manufacturer's recommendations (TranscriptAid T7 High Yield Transcription Kit, ThermoFisher) as follows: ATP / CTP / GTP / m1ψTP: 5mM each SmartCap (SC101, ST Pharm): 4mM Linear template DNA: 1 μg of plasmid or 0.5 μg of PCR product T7 RNA polymerase enzyme mix: 2 μl

[0113] IVT was performed in a 20 μl reaction by incubation at 37°C for 2 hours. Template DNA was removed with 2 units of DNase I (ThermoFisher) for 15 minutes at 37°C, followed by column purification (Monarch RNA Cleanup Kit, New England Biolabs).

[0114] The IVT products of the RSV F mutant B strain gene and the RSV F mutant A strain gene were analyzed by agarose gel, and their 2-kn-long mRNA products were detected. Here, after IVT from DNA templates of RSV F mutant B strain and RSV F mutant A strain mRNA, 100 ng of mRNA was electrophoresed on 1% agarose E-GEL EX in an E-Gel Power Snap Electrophoresis Device (ThermoFisher), as shown in Figure 1 (bottom).

[0115] Example 3 - Transfection and Western Blot 1 μg of each mRNA was transfected into 293FT cells or SJCRH30 (CRL-2061, ATCC) in 12-well plates using 2 μl or 4 μl of Lipofectamine MesseangerMax (ThermoFisher) at a 1:2 ratio according to the manufacturer's protocol. Cell lysates were prepared in NP-40 lysis buffer (150 mM sodium chloride / 1% NP-40 / 50 mM Tris (pH 8.0)) 24 hours after transfection.

[0116] Mouse monoclonal anti-RSV-F antibody (NB110-37246, Noivus Biologicals) was used for Western blotting, and HRP-conjugated goat anti-mouse IgG antibody (115-035-062, Jackson ImmunoResearch) and SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoScientific) were used for protein detection.

[0117] Expression of RSV F mutant proteins was detected in 293FT cells transfected with individual mRNAs (Fig. 2). 1 μg of RSV mRNA was individually transfected into 293FT cells. Cell lysates were subjected to Western blotting to detect RSV F mutants of strains B and A (lanes 2 and 4), as shown in Fig. 2. Lane 1: Negative control.

[0118] Example 4 - Immunogenicity studies of RSV vaccine compositions This study was designed to test the immunogenicity in mice of the RSV vaccine composition of the present disclosure.

[0119] Mice were immunized intramuscularly (IM) with the RSV vaccine compositions of the present disclosure (RSV F mutant B strain and RSV F mutant A strain). The vaccine compositions of the present disclosure were chemically modified or unmodified. A total of two immunizations were performed at 3-week intervals (i.e., week 0 and week 3), and serum was collected after each immunization until week 7 (weeks 2, 5, and 7) (FIG. 3). Serum IgG titers specific for the Pre-F protein in serum collected at weeks 2, 5, and 7 were determined by ELISA (FIG. 4). Neutralizing titers of pooled sera from weeks 2, 5, and 7 against RSV A and B strains were determined separately by a plaque-based neutralization assay (FIG. 5). To detect T cell responses to the RSV vaccine in vaccinated mice, spleens were harvested from immunized and RSV-challenged (4 days) mice and RSV F was detected. 85-93 The cells were stained with tetramer (Figure 6). 85-93The percentage of CD8+ T cells stained with tetramer was determined by flow cytometry analysis (Fig. 6).

[0120] Example 5 - Viral Challenge Study of RSV Vaccine Compositions This study was designed to evaluate the protective efficacy of RSV vaccines against RSV subtype A and subtype B infection in mice after immunization with the appropriate dose and formulation described in the immunogenicity study. Mice were immunized twice at weeks 0 and 3 and divided into two groups, and at week 8, each group was infected separately with either the RSV A2 or RSV B1 strain. In vivo lung virus titers were monitored 4 days post-infection. Virus titers isolated from harvested lungs were determined by plaque assay for RSV A2 and Q-RT-PCR for RSV B1 (Figure 7).

[0121] Example 6 - DNA template used for in vitro transcription and protein expression of hMPV F gene (see Figure 8) The DNA construct used for hPMV F protein mRNA was prepared as shown in FIG. 8, top.

[0122] IVT (FIG. 8, bottom left): hMPV F protein mRNA was produced as described in FIG. 8, bottom left.

[0123] Western blot (Figure 8, bottom right): Detection of hMPV F protein from mRNA was performed as described above for RSV F protein. Mouse monoclonal anti-FLAG antibody (AB_1957945, Invitrogen) was used against FLAG-tagged hMPV-F protein. As shown in Figure 8, hMPV F protein expression was detected in 293FT cells transfected with mRNA containing a FLAG tag at the 3' end of the hMPV-F ORF.

[0124] Example 7 - Immunogenicity studies of RSV and hMPV vaccine compositions This study was designed to test the immunogenicity in mice of the RSV vaccine composition or RSV and hMPV vaccine composition of the present disclosure.

[0125] Mice were immunized intramuscularly (IM) or intradermally (ID) with the RSV vaccine compositions of the present disclosure (RSV F mutant B strain and RSV F mutant A strain) or the RSV and hMPV vaccine compositions of the present disclosure (RSV F mutant B strain, RSV F mutant A strain, and hMPV F). The vaccine compositions of the present disclosure may be chemically modified or unmodified. A total of four immunizations were administered at 3-week intervals (i.e., week 0 and week 3), with serum collected after each immunization until week 8. Serum antibody titers against the RSV F mutant B strain, RSV F mutant A strain, and hMPV F were determined by ELISA. Sera collected from each mouse between weeks 10 and 16 were randomly pooled and used in virus neutralization assays. Purified IgG antibodies were used for immunoelectron microscopy, antibody affinity testing, and in vitro protection assays. The spleens of immunized mice were removed and used to measure T cell responses to various cytokines, including TNFα, INF-γ, and IL-2, following peptide stimulation. Doses and formulations will be selected for virus challenge studies.

[0126] Example 8 - Viral challenge studies of RSV and hMPV vaccine compositions This study is designed to evaluate the protective efficacy of RSV vaccines or RSV and hMPV vaccines against RSV and / or hMPV infection in mice after immunization with appropriate doses and combinations determined in immunogenicity studies. Mice will be immunized twice, at weeks 0 and 3, and immunized mice will be infected with RSV and / or hMPV at week 6. Phenotypes (weight loss, fever, and survival) will be monitored for two weeks post-infection. Viral titers will be determined in serum collected at weeks 1 and 2 post-infection. Mice will be sacrificed and examined for pathology.

Claims

1. A respiratory syncytial virus (RSV) vaccine composition comprising a messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding an RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO:

1.

2. 2. The RSV vaccine composition of claim 1, wherein the ORF encoding an RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO:

2.

3. The mRNA comprising an ORF encoding an RSV F mutant B strain protein has the following structure: 5'UTR-ORF encoding RSV F mutant B strain protein-3'UTR-poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the 3. The RSV vaccine composition of claim 2, wherein the ORF encoding an RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO:

2.

4. 4. The RSV vaccine composition of claim 3, wherein the poly(A) tail has a length of 50 to 250 nucleotides.

5. The RSV vaccine composition of claim 3, wherein the mRNA having the structure of 5'UTR-ORF encoding RSV F mutant B strain protein-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

3.

6. The RSV vaccine composition of claim 3, wherein the mRNA having the structure of 5'UTR-ORF encoding RSV F mutant B strain protein-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO:

3.

7. 10. The RSV vaccine composition of claim 1, further comprising a pharmaceutically acceptable carrier.

8. 8. The RSV vaccine composition of claim 7, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

9. 10. A method for inducing an immune response against respiratory syncytial virus (RSV), comprising administering an effective amount of the RSV vaccine composition of claim 1 to a subject in need thereof.

10. 10. The RSV vaccine composition of claim 1, further comprising an mRNA comprising an ORF encoding an RSV F mutant A strain protein having the amino acid sequence of SEQ ID NO:

5.

11. 11. The RSV vaccine composition of claim 10, wherein the ORF encoding an RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO:

6.

12. The mRNA comprising an ORF encoding an RSV F mutant A strain protein has the following structure: 5'UTR-ORF encoding RSV F mutant A strain protein-3'UTR-poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the 12. The RSV vaccine composition of claim 11, wherein the ORF encoding an RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO:

6.

13. 13. The RSV vaccine composition of claim 12, wherein the poly(A) tail has a length of 50 to 250 nucleotides.

14. The RSV vaccine composition of claim 12, wherein the mRNA having the structure of 5'UTR-ORF encoding RSV F mutant A strain protein-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

7.

15. The RSV vaccine composition of claim 12, wherein the mRNA having the structure of 5'UTR-ORF encoding RSV F mutant A strain protein-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO:

7.

16. 11. The RSV vaccine composition of claim 10, further comprising a pharmaceutically acceptable carrier.

17. 17. The RSV vaccine composition of claim 16, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

18. 11. A method for inducing an immune response against respiratory syncytial virus (RSV), comprising administering an effective amount of the RSV vaccine composition of claim 10 to a subject in need thereof.

19. 1. A respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccine composition comprising: a messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding an RSV F mutant A strain protein having the amino acid sequence of SEQ ID NO:5; an mRNA comprising an ORF encoding an RSV F mutant B strain protein having the amino acid sequence of SEQ ID NO:1; an mRNA containing an ORF encoding a hMPV F protein having the amino acid sequence of SEQ ID NO:9; 1. A RSV and hMPV vaccine composition comprising:

20. the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO:6; the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO:2; the ORF encoding the hMPV F protein has the nucleotide sequence of SEQ ID NO: 10; 20. The RSV and hMPV vaccine composition of claim 19.

21. (i) the mRNA comprising an ORF encoding an RSV F mutant A strain protein has the following structure: 5'UTR-ORF encoding RSV F mutant A strain protein-3'UTR-poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the the ORF encoding the RSV F mutant A strain protein has the nucleotide sequence of SEQ ID NO:6; (ii) the mRNA comprising an ORF encoding an RSV F mutant B strain protein has the following structure: 5'UTR-ORF encoding RSV F mutant B strain protein-3'UTR-poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the the ORF encoding the RSV F mutant B strain protein has the nucleotide sequence of SEQ ID NO:2; (iii) the mRNA comprising an ORF encoding the hMPV F protein has the following structure: 5'UTR-ORF encoding hMPV F protein-3'UTR-poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the the ORF encoding the hMPV F protein has the nucleotide sequence of SEQ ID NO: 10; 21. The RSV and hMPV vaccine composition of claim 20.

22. 22. The RSV and hMPV vaccine composition of claim 21, wherein the poly(A) tail has a length of 50 to 250 nucleotides.

23. (i) the mRNA having the structure of 5'UTR-ORF encoding the RSV F mutant A strain protein-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7; (ii) the mRNA having the structure of 5'UTR-ORF encoding the RSV F mutant B strain protein-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 3; (iii) the mRNA having the structure of 5'UTR-ORF encoding hMPV F protein-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 11; 22. The RSV and hMPV vaccine composition of claim 21.

24. (i) the mRNA having the structure of 5'UTR-ORF encoding RSV F mutant A strain protein-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO:7; (ii) the mRNA having the structure of 5'UTR-ORF encoding RSV F mutant B strain protein-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO:3; (iii) the mRNA having the structure of 5'UTR-ORF encoding hMPV F protein-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO: 11; 22. The RSV and hMPV vaccine composition of claim 21.

25. 20. The RSV and hMPV vaccine composition of claim 19, further comprising a pharmaceutically acceptable carrier.

26. 26. The RSV and hMPV vaccine composition of claim 25, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

27. A method for inducing an immune response against respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), comprising administering an effective amount of the RSV and hMPV vaccine composition of claim 19 to a subject in need thereof.

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