Combination respiratory tract mRNA vaccine

Abstract

The present disclosure provides compositions comprising at least two messenger RNAs (mRNA) consisting of a human respiratory syncytial virus (hRSV or RSV) F protein antigen, a human metapneumovirus (hMPV) F protein antigen, and / or a human parainfluenza virus 3 (hPIV3 or PIV3) F protein antigen; and methods of eliciting an immune response by administering the composition.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 465,389, filed May 10, 2023, and European Provisional Patent Application Serial No. 24315228.7, filed May 9, 2024, the disclosures of which are hereby incorporated in their entirety by reference. Background Technology

[0002] Human respiratory syncytial virus (hRSV or RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3 or PIV3) are respiratory pathogens associated with high morbidity and mortality rates in infants, children, and the elderly, with a combined disease burden exceeding that of influenza. Because there are currently no available vaccines or specific treatments, there is an unmet need for safe, effective, and convenient mRNA combination vaccines to protect individuals from these pathogens. Summary of the Invention

[0003] In one aspect, this disclosure provides compositions comprising at least two messenger RNAs (mRNAs), wherein the at least two mRNAs comprise an open reading frame (ORF) encoding a recombinant F protein antigenic polypeptide, selected from the group consisting of: (i) a first mRNA encoding a human respiratory syncytial virus (hRSV) F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; (ii) a second mRNA encoding a human metapneumovirus (hMPV) F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a third mRNA encoding a human parainfluenza virus 3 (hPIV3) F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0004] In some embodiments, the composition comprises a first mRNA encoding the amino acid sequence of SEQ ID NO: 1; and a second mRNA encoding the amino acid sequence of SEQ ID NO: 2.

[0005] In some embodiments, the composition comprises a first mRNA encoding the amino acid sequence of SEQ ID NO: 1; and a third mRNA encoding the amino acid sequence of SEQ ID NO: 3.

[0006] In some embodiments, the composition comprises a second mRNA encoding the amino acid sequence of SEQ ID NO: 2; and a third mRNA encoding the amino acid sequence of SEQ ID NO: 3.

[0007] In some embodiments, the composition comprises at least three mRNAs, including: a first mRNA encoding the amino acid sequence of SEQ ID NO: 1; a second mRNA encoding the amino acid sequence of SEQ ID NO: 2; and a third mRNA encoding the amino acid sequence of SEQ ID NO: 3.

[0008] In some embodiments, the composition comprises three mRNAs, including: a first mRNA encoding the amino acid sequence of SEQ ID NO: 1; a second mRNA encoding the amino acid sequence of SEQ ID NO: 2; and a third mRNA encoding the amino acid sequence of SEQ ID NO: 3.

[0009] In some embodiments, the hRSV F protein contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

[0010] In some embodiments, the hMPV F protein contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.

[0011] In some embodiments, the hPIV3 F protein contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 3.

[0012] In some embodiments, the first mRNA comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO: 4.

[0013] In some embodiments, the second mRNA comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 5.

[0014] In some embodiments, the third mRNA comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 6.

[0015] In some embodiments, at least one of these recombinant F protein antigenic peptides is a pre-fusion protein.

[0016] In some embodiments, at least one of these mRNAs contains a codon-optimized ORF.

[0017] In some embodiments, at least one of these mRNAs comprises at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and at least one polyadenylated (poly(A)) sequence.

[0018] In some embodiments, at least one of these mRNAs comprises a 5' UTR containing a nucleic acid sequence having at least 80% identity with SEQ ID NO: 7.

[0019] In some embodiments, at least one of these mRNAs comprises a 3' UTR containing a nucleic acid sequence having at least 80% identity with SEQ ID NO: 8.

[0020] In some embodiments, at least one of these mRNAs contains at least one chemical modification.

[0021] 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 at least one of these mRNAs are chemically modified.

[0022] 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 at least one of these ORFs are chemically modified.

[0023] In some embodiments, the chemical modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-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-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0024] In some embodiments, the chemical modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0025] In some embodiments, the chemical modification is N1-methylpseuuridine.

[0026] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:1 (w / w).

[0027] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:2:1 (w / w).

[0028] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:3:1 (w / w).

[0029] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:5:1 (w / w).

[0030] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present in a ratio (w / w) of about 1:1:1 to about 1:10:1.

[0031] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:1:0.1 to about 1:1:1 (w / w).

[0032] In some embodiments, the ratio is expressed in micrograms (µg).

[0033] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 1 microgram of the second mRNA to about 1 microgram of the third mRNA.

[0034] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 2 micrograms of the second mRNA to about 1 microgram of the third mRNA.

[0035] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 3 micrograms of the second mRNA to about 1 microgram of the third mRNA.

[0036] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 5 micrograms of the second mRNA to about 1 microgram of the third mRNA.

[0037] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

[0038] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

[0039] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.1 micrograms of the third mRNA.

[0040] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1 microgram of the second mRNA to about 0.5 micrograms of the third mRNA.

[0041] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 2.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

[0042] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are not covalently linked to each other.

[0043] In some embodiments, one or more of the first mRNA, the second mRNA, and the third mRNA are covalently linked to each other.

[0044] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are each formulated into separate nanoparticles (LNPs).

[0045] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are formulated into the same LNP.

[0046] In some embodiments, the LNP contains at least one cationic lipid.

[0047] In some embodiments, the cationic lipid is biodegradable.

[0048] In some embodiments, the cationic lipid is not biodegradable.

[0049] In some embodiments, the cationic lipid is cleavable.

[0050] In some embodiments, the cationic lipid is not cleavable.

[0051] In some 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, GL-HEPES-E3-E12-DS-3-E14 and IM-001.

[0052] In some embodiments, the cationic lipid is cKK-E10.

[0053] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

[0054] In some embodiments, the cationic lipid is IM-001.

[0055] In some embodiments, the LNP further comprises polyethylene glycol (PEG) conjugated (PEGylated) lipids, cholesterol-based lipids, and auxiliary lipids.

[0056] In some embodiments, the LNP comprises: 35% to 55% cationic lipids; 0.25% to 2.75% polyethylene glycol (PEG) conjugated (PEGylated) lipids; 20% to 45% cholesterol-based lipids; and 5% to 35% auxiliary lipids, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0057] In some embodiments, the LNP comprises: 40% cationic lipids; 1.5% PEGylated lipids; 28.5% cholesterol-based lipids; and 30% auxiliary lipids.

[0058] In some embodiments, the PEGylated lipid is dimyristic-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0059] In some embodiments, the cholesterol-based lipid is cholesterol.

[0060] In some embodiments, the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0061] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-4-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio.

[0062] In some embodiments, the LNP comprises: 40% cKK-E10 in molar ratio; 1.5% DMG-PEG2000 in molar ratio; 28.5% cholesterol in molar ratio; and 30% DOPE in molar ratio.

[0063] In some embodiments, the LNP comprises: 40% IM-001; 1.5% DMG-PEG2000; 28.5% cholesterol; and 30% DOPE.

[0064] In some embodiments, the average diameter of the LNP is 30 nm to 200 nm.

[0065] In some embodiments, the average diameter of the LNP is 80 nm to 150 nm.

[0066] In some embodiments, at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Clustered (A) tail.

[0067] In one aspect, this disclosure provides a composition comprising at least two mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0068] In one aspect, this disclosure provides a composition comprising at least two mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0069] In one aspect, this disclosure provides a composition comprising at least two mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0070] In one aspect, this disclosure provides a composition comprising at least two mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10, IM-001, OF-02 or cKK-E10 in a molar ratio of 35% to 55%.

[0071] On the other hand, this disclosure provides a method for inducing an immune response to hRSV or protecting a subject from hRSV infection, the method comprising administering the above-described composition to the subject.

[0072] On the other hand, this disclosure provides a method for inducing an immune response to hMPV or protecting a subject from hMPV infection, the method comprising administering the above-described composition to the subject.

[0073] On the other hand, this disclosure provides a method for inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the method comprising administering the above-described composition to the subject.

[0074] In some embodiments, after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hRSV neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO:1.

[0075] In some embodiments, after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hMPV neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO:2.

[0076] In some embodiments, after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hPIV3 neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hPIV3 F protein antigen of SEQ ID NO:3.

[0077] In some embodiments, after administration of the composition, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies relative to subjects administered the protein hRSV vaccine.

[0078] In some embodiments, after administration of the composition, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies relative to a subject administered the protein hMPV vaccine.

[0079] In some embodiments, after administration of the composition, the subject has a comparable serum concentration of anti-hPIV3 neutralizing antibodies relative to a subject administered the protein hPIV3 vaccine.

[0080] In some embodiments, the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hRSV.

[0081] In some embodiments, the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV.

[0082] In some embodiments, the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hPIV3.

[0083] In another aspect, this disclosure provides a composition for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering the composition to the subject.

[0084] In another aspect, this disclosure provides a composition for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering the composition to the subject.

[0085] In another aspect, this disclosure provides a composition for use in inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the use comprising administering the composition to the subject.

[0086] On the other hand, this disclosure provides the use of the composition in the manufacture of medicaments for inducing an immune response to hRSV or protecting subjects from hRSV infection.

[0087] On the other hand, this disclosure provides the use of the composition in the manufacture of medicaments for inducing an immune response to hMPV or protecting subjects from hMPV infection.

[0088] On the other hand, this disclosure provides the use of the composition in the manufacture of medicaments for inducing an immune response to hPIV3 or protecting subjects from hPIV3 infection.

[0089] In one aspect, this disclosure provides a composition comprising three mRNAs, wherein: (i) a first mRNA encoding an hRSV F protein antigen containing the amino acid sequence of SEQ ID NO: 1; (ii) a second mRNA encoding a human metapneumovirus (hMPV) F protein antigen containing the amino acid sequence of SEQ ID NO: 2; and (iii) a third mRNA encoding a human parainfluenza virus 3 (hPIV3) F protein antigen containing the amino acid sequence of SEQ ID NO: 3; wherein the first mRNA, the second mRNA, and the third mRNA are formulated into the same LNP, the same LNP comprising: 40% of 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; 1.5% of DMG-PEG2000; 28.5% of cholesterol; and 30% of DOPE.

[0090] In one aspect, this disclosure provides a composition comprising three mRNAs, wherein: (i) a first mRNA encodes a human respiratory syncytial virus (hRSV) F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; (ii) a second mRNA encodes a human metapneumovirus (hMPV) F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a third mRNA encodes a human parainfluenza virus 3 (hPIV3) F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0091] In one aspect, this disclosure provides a composition comprising a first messenger RNA (mRNA) and a second mRNA, wherein: (i) the first mRNA encodes a human respiratory syncytial virus (hRSV) F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes a human metapneumovirus (hMPV) F protein antigen comprising the amino acid sequence of SEQ ID NO: 2.

[0092] In some embodiments, the first mRNA and the second mRNA are present in a ratio of approximately 1:1 (w / w).

[0093] In some embodiments, the first mRNA and the second mRNA are present in a ratio of approximately 1:2 (w / w).

[0094] In some embodiments, the first mRNA and the second mRNA are present in a ratio of approximately 1:3 (w / w).

[0095] In some embodiments, the first mRNA and the second mRNA are present in a ratio of approximately 1:5 (w / w).

[0096] In some embodiments, the first mRNA and the second mRNA are present in a ratio of about 1:1 to about 1:10 (w / w).

[0097] In some embodiments, the ratio of the first mRNA to the second mRNA is expressed in micrograms (µg).

[0098] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA.

[0099] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1.5 micrograms of the second mRNA.

[0100] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1 microgram of the second mRNA.

[0101] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 2.5 micrograms of the second mRNA.

[0102] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 1 microgram of the second mRNA.

[0103] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 2 micrograms of the second mRNA.

[0104] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 3 micrograms of the second mRNA.

[0105] In some embodiments, the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 5 micrograms of the second mRNA.

[0106] In some embodiments, the first mRNA and the second mRNA are each formulated into separate LNPs.

[0107] In some embodiments, the first mRNA and the second mRNA are formulated into the same LNP.

[0108] In some embodiments, the LNP contains at least one cationic lipid.

[0109] In some embodiments, the cationic lipid is biodegradable.

[0110] In some embodiments, the cationic lipid is not biodegradable.

[0111] In some embodiments, the cationic lipid is cleavable.

[0112] In some embodiments, the cationic lipid is not cleavable.

[0113] In some 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, GL-HEPES-E3-E12-DS-3-E14 and IM-001.

[0114] In some embodiments, the cationic lipid is cKK-E10.

[0115] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

[0116] In some embodiments, the cationic lipid is IM-001.

[0117] In some embodiments, the LNP further comprises polyethylene glycol (PEG) conjugated (PEGylated) lipids, cholesterol-based lipids, and auxiliary lipids.

[0118] In some embodiments, the LNP comprises: 35% to 55% cationic lipids; 0.25% to 2.75% polyethylene glycol (PEG) conjugated (PEGylated) lipids; 20% to 45% cholesterol-based lipids; and 5% to 35% auxiliary lipids, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0119] In some embodiments, the LNP comprises: 40% cationic lipids; 1.5% PEGylated lipids; 28.5% cholesterol-based lipids; and 30% auxiliary lipids.

[0120] In some embodiments, the PEGylated lipid is dimyristic-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0121] In some embodiments, the cholesterol-based lipid is cholesterol.

[0122] In some embodiments, the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0123] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-4-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio.

[0124] In some embodiments, the LNP comprises: 40% cKK-E10 in molar ratio; 1.5% DMG-PEG2000 in molar ratio; 28.5% cholesterol in molar ratio; and 30% DOPE in molar ratio.

[0125] In some embodiments, the LNP comprises: 40% IM-001; 1.5% DMG-PEG2000; 28.5% cholesterol; and 30% DOPE.

[0126] In some embodiments, the average diameter of the LNP is 30 nm to 200 nm.

[0127] In some embodiments, the average diameter of the LNP is 80 nm to 150 nm.

[0128] On the other hand, this disclosure provides a method for inducing an immune response to hRSV or protecting a subject from hRSV infection, the method comprising administering the above-described composition to the subject.

[0129] On the other hand, this disclosure provides a method for inducing an immune response to hMPV or protecting a subject from hMPV infection, the method comprising administering the above-described composition to the subject.

[0130] In some embodiments, after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hRSV neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO:1.

[0131] In some embodiments, after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hMPV neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO:2.

[0132] In some embodiments, after administration of the composition, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies relative to subjects administered the protein hRSV vaccine.

[0133] In some embodiments, after administration of the composition, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies relative to a subject administered the protein hMPV vaccine.

[0134] In some embodiments, the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hRSV.

[0135] In some embodiments, the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV.

[0136] In another aspect, this disclosure provides a composition for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering the composition to the subject.

[0137] In another aspect, this disclosure provides a composition for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering the composition to the subject.

[0138] On the other hand, this disclosure provides the use of the composition in the manufacture of medicaments for inducing an immune response to hRSV or protecting subjects from hRSV infection.

[0139] On the other hand, this disclosure provides the use of the composition in the manufacture of medicaments for inducing an immune response to hMPV or protecting subjects from hMPV infection. Attached Figure Description

[0140] The foregoing descriptions and other features and advantages of this disclosure will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings.

[0141] Figures 1A-1F Protein expression in human skeletal muscle (hSKM) cells transfected with monovalent mRNA-LNP formulations of hRSV, hPIV3, or hMPV (dosage range from 0.088 µg to 3 µg) and trivalent mRNA-LNP formulations of hRSV, hPIV3, and hMPV (dosage range from 0.088 µg to 3 µg, in a 1:1:1 ratio). The mRNA constructs were formulated with LNP OF-02. 24 hours post-transfection, hSKM cells were harvested and subjected to a pre-fusion binding monoclonal antibody (…). Figure 1A , 1C and 1E) or common region binding monoclonal antibodies ( Figure 1B , 1D The cells were stained with 1F and fluorescently labeled secondary antibodies. Fluorescence was quantified by flow cytometry and displayed as integrated mean fluorescence intensity (iMFI).

[0142] Figures 2A-2F Immunogenicity in mice following vaccination with the following formulations was characterized: a trivalent mRNA-LNP co-formulation (each dose containing 0.5 µg pre-fusion hRSV mRNA, 0.5 µg pre-fusion hPIV3 mRNA, and 0.5 µg pre-fusion hMPV mRNA), a mixture of three monovalent mRNA-LNP formulations (containing 0.5 µg / dose of pre-fusion hRSV mRNA, 0.5 µg / dose of pre-fusion hPIV3 mRNA, and 0.5 µg / dose of pre-fusion hMPV mRNA), or a monovalent mRNA-LNP formulation (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). The mRNA constructs were formulated with LNP OF-02. All formulations were diluted in trehalose. Mice were boosted 21 days after primacy using the same protocol, and blood was collected before the first immunization (day 0) and 2 weeks after the second immunization (day 35). Use the encapsulated hRSV before fusion ( Figure 2A ), before fusion hPIV3 ( Figure 2C ) or before fusion hMPV ( Figure 2E The antibody titer was determined by ELISA using a plate containing a green fluorescent protein (GFP) reporter. The titer was determined by ELISA using hRSV (a plate containing GFP reporter). Figure 2B ), hPIV3 ( Figure 2D ) or hMPV ( Figure 2F The titer of neutralizing antibodies was determined by micro-neutralization. Day 0 samples were used as the pool in the micro-neutralization assay. The dashed line indicates the lower limit of quantitation. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA with a mixed model was used. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0143] Figures 3A-3FImmunogenicity in mice following vaccination with a mixture of pre-fusion recombinant proteins of hRSV, hPIV3, and hMPV, or monovalent pre-fusion recombinant proteins of hRSV, hPIV3, or hMPV, was characterized. Intramuscular vaccination was administered to mice using either a mixture adjuvanted with Alhydrogel® 2% (aluminum hydroxide gel) containing 0.5 µg of each of the pre-fusion recombinant proteins of hRSV, hPIV3, and hMPV, or monovalent pre-fusion recombinant proteins adjuvanted with Alhydrogel® containing 0.5 µg of pre-fusion hRSV, pre-fusion hPIV3, or pre-fusion hMPV protein per dose. All formulations were diluted with trehalose. Mice were boosted with the same regimen 21 days after primacy, and blood was collected before the first immunization (day 0) and 2 weeks after the second immunization (day 35). Pre-fusion hRSV (…) was used… Figure 3A ), hPIV3 ( Figure 3B ) or hMPV ( Figure 3C The titer of the binding antibody was determined by ELISA using a plate containing a GFP reporter (hRSV). Figure 3D ), hPIV3 ( Figure 3E ) or hMPV ( Figure 3F The titer of neutralizing antibodies was determined by micro-neutralization. Day 0 samples were run as a pool in the micro-neutralization assay. Dashed lines indicate the lower limit of quantitation. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0144] Figures 4A-4F Immunogenicity in mice was characterized by increasing the hMPV dose in a mixture of monovalent hRSV, hMPV, and hPIV3 mRNA-LNP formulations in LNP OF-02. Mice were immunized intramuscularly with a mixture of pre-fusion hRSV:hMPV:hPIV3 mRNA in a ratio of 1:1:1, 1:2:1, or 1:5:1, or with 0.5 µg / dose of monovalent hMPV mRNA. All formulations were diluted in trehalose. Mice were boosted with the same protocol 21 days after primacy, and blood was collected before the first immunization (day 0) and 2 weeks after the second immunization (day 35). Pre-fusion hRSV (… Figure 4A ), hPIV3 ( Figure 4B ) or hMPV ( Figure 4CThe titer of the binding antibody was determined by ELISA using a plate containing a GFP reporter (hRSV). Figure 4D ), hPIV3 ( Figure 4E ) or hMPV ( Figure 4F The titer of neutralizing antibodies was determined by micro-neutralization. Day 0 samples were run as a pool in the micro-neutralization assay. Dashed lines indicate the lower limit of quantitation. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0145] Figures 5A-5F Immunogenicity in mice was depicted after reducing the hPIV3 dose in a mixture of hRSV, hMPV, and hPIV3 monovalent mRNAs from LNP OF-02 formulations. Mice were immunized intramuscularly with the following mixtures: a 1:1:1 mixture of pre-fusion hRSV:hMPV:hPIV3 mRNA (i.e., 0.5 µg pre-fusion hRSV mRNA, 0.5 µg pre-fusion hMPV, and 0.5 µg hPIV3 mRNA), a 1:1:0.2 mixture of pre-fusion hRSV:hMPV:hPIV3 mRNA (i.e., 0.5 µg pre-fusion hRSV mRNA, 0.5 µg pre-fusion hMPV, and 0.1 µg hPIV3 mRNA), or 0.1 µg / dose of monovalent hPIV3 mRNA. All formulations were diluted in trehalose. Mice were boosted with the same protocol 21 days after primary immunization, and blood was collected before the first immunization (day 0) and 2 weeks after the second immunization (day 35). Pre-fusion hRSV (coated with...) was used. Figure 5A ), hPIV3 ( Figure 5B ) or hMPV ( Figure 5C The titer of the binding antibody was determined by ELISA using a plate containing a GFP reporter (hRSV). Figure 5D ), hPIV3 ( Figure 5E ) or hMPV ( Figure 5F The titer of neutralizing antibodies was determined by micro-neutralization. Day 0 samples were run as a pool in the micro-neutralization assay. Dashed lines indicate the lower limit of quantitation. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0146] Figures 6A-6F Immunogenicity in mice was characterized by increasing the hMPV dose in the trivalent hRSV:hMPV:hPIV3 co-preparation tested in LNP OF-02. Mice were immunized intramuscularly with the following co-preparations: 1:1:1, 1:2:1, or 1:5:1 of pre-fusion hRSV:hMPV:hPIV3 mRNA, or monovalent preparations (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). All preparations were diluted in trehalose. Mice were boosted with the same protocol 21 days after primacy, and blood was collected before the first immunization (day 0) and 2 weeks after the second immunization (day 35). Pre-fusion hRSV (…) was used… Figure 6A ), hPIV3 ( Figure 6C ) or hMPV ( Figure 6E The titer of the binding antibody was determined by ELISA using a plate containing a GFP reporter (hRSV). Figure 6B ), hPIV3 ( Figure 6D ) or hMPV ( Figure 6F The titer of neutralizing antibodies was determined by micro-neutralization. Day 0 samples were run as a pool in the micro-neutralization assay. Dashed lines indicate the lower limit of quantitation. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0147] Figures 7A-7FImmunogenicity in mice was depicted after increasing the hMPV dose in the trivalent hRSV:hMPV:hPIV3 co-preparation tested in LNP cKK-E10. Mice were immunized intramuscularly with either a 1:2:1 or 1:5:1 co-preparation of pre-fusion hRSV:hMPV:hPIV3 mRNA, or a monovalent preparation (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). All preparations were diluted in trehalose. Mice were boosted with the same protocol 21 days after primacy, and blood was collected before the first immunization (day 0) and 2 weeks after the second immunization (day 35). Pre-fusion hRSV (…) was used… Figure 7A ), hPIV3 ( Figure 7C ) or hMPV ( Figure 7E The titer of the binding antibody was determined by ELISA using a plate containing a GFP reporter (hRSV). Figure 7B ), hPIV3 ( Figure 7D ) or hMPV ( Figure 7F The titer of neutralizing antibodies was determined by micro-neutralization. Day 0 samples were run as a pool in the micro-neutralization assay. Dashed lines indicate the lower limit of quantitation. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0148] Figures 8A-8C This describes the hRSV:hMPV:hPIV3 mRNA co-preparations (1:1:1), (1:3:1), and (monovalent preparations of hRSV, hMPV, and hPIV3) as tested with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10, respectively). Figure 8A hMPV ( Figure 8B ) and hPIV3 ( Figure 8CWestern blot analysis of protein expression. HeLa cells were seeded overnight in 24-well plates at 75,000 cells / well and transfected the following day with a specified mRNA formulation at 1 µg / million cells. Twenty-four hours post-transfection, cells were lysed, and the clear lysates were run on SDS-PAGE at an 8%–16% gradient. After transfer to nitrocellulose membranes, hRSV, hMPV, and hPIV3 protein expression were detected using antibodies CR9501, SAN27K-37, or SAN52-36, respectively. All formulations containing hRSV, hMPV, or hPIV3 mRNA were positive for the expression of the corresponding proteins.

[0149] Figures 9A-9F Immunogenicity in mice immunized with trivalent hRSV:hMPV:hPIV3 co-preparations tested in LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10, with protein expression confirmed in Figure 8, is depicted. Mice were immunized intramuscularly in the following groups: 1:1:1 co-formulations of hRSV:hMPV:hPIV3 mRNA prepared with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10 (each dose containing 0.5 µg pre-fusion hRSV mRNA, 0.5 µg pre-fusion hMPV mRNA, and 0.5 µg pre-fusion hPIV3 mRNA); 1:3:1 co-formulations of hRSV:hMPV:hPIV3 mRNA prepared with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10 (each dose containing 0.5 µg pre-fusion hRSV mRNA, 1.5 µg pre-fusion hMPV mRNA, and 0.5 µg pre-fusion hPIV3 mRNA); or LNP... 0.5 µg / dose of monovalent RSV, hMPV, or hPIV3 mRNA prepared with OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10. Dilute the preparations with phosphate-buffered saline (PBS) to the specified concentration. Mice were boosted 21 days after primary immunization using the same protocol, with blood collected before the first immunization (day 0), before the second immunization (day 21), and 2 weeks after the second immunization (day 35). Use pre-fusion hMPV (… Figure 9B ), hPIV3 ( Figure 9D ) or hRSV ( Figure 9F The titer of the binding antibody was determined by ELISA using plates containing GFP reporter hMPV ( Figure 9A ), hPIV3 ( Figure 9C ) or hRSV ( Figure 9E The neutralizing antibody titer was determined by trace neutralization. The dashed line represents the lower limit of quantitation. In all assays, all day 0 samples were below the lower limit of quantitation. Samples from groups immunized with monovalent formulations were tested only against the corresponding target. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0150] Figure 10 Neutralizing antibody titers of hRSV, hMPV, and hPIV3 mRNA constructs diluted with PBS were plotted against those of hRSV, hMPV, and hPIV3 mRNA constructs diluted with trehalose. Mice were immunized intramuscularly with a 1:1:1 co-preparation of hRSV:hMPV:hPIV3 mRNA formulated with LNP OF-02 (each dose containing 0.5 µg pre-fusion hRSV mRNA, 0.5 µg pre-fusion hMPV mRNA, and 0.5 µg pre-fusion hPIV3 mRNA) diluted to this concentration with trehalose or PBS. Mice were boosted 21 days after priming using the same protocol, and blood was collected before the first immunization (day 0), before the second immunization (day 21), and 2 weeks after the second immunization (day 35). Neutralizing antibody titers were determined by micro-neutralization against hMPV, hPIV3, or hRSV expressing the GFP reporter. The dashed line represents the lower limit of quantitation. In all assays, all day 0 samples were below the lower limit of quantitation. The error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0151] Figures 11A-11DImmunogenicity in mice immunized with bivalent hRSV:hMPV co-formulations tested in LNP OF-02, LNP GL-HEPES-E3-E12-DS-4-E10, or LNPIM-001 was depicted. Mice were immunized with either a 1:1 co-formulation of hRSV:hMPV mRNA prepared with LNP OF-02, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP IM-001 (each dose containing 0.5 µg of pre-fusion hRSV mRNA and 0.5 µg of pre-fusion hMPV mRNA); or 0.5 µg / dose of monovalent hRSV or hMPV mRNA prepared with LNP OF-02, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP IM-001. All formulations were diluted in PBS. Mice were boosted with the same protocol 21 days after primary immunization. Blood was collected before the first immunization (day 0), before the second immunization (day 21), and 2 weeks after the second immunization (day 35). Pre-fusion hRSV (coated with...) was used... Figure 11B ) or hMPV ( Figure 9D The titer of the binding antibody was determined by ELISA using a plate containing a GFP reporter (hRSV). Figure 11A ) or hMPV ( Figure 11C The neutralizing antibody titer was determined by trace neutralization. The dashed line represents the lower limit of quantitation. In all assays, all day 0 samples were below the lower limit of quantitation. Samples from groups immunized with monovalent formulations were tested only against the corresponding target. Error bars represent one geometric standard deviation from the geometric mean. A two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001.

[0152] Figure 12A-12BThe immunogenicity of hRSV, hMPV, and hPIV3 in mice immunized with a bivalent hRSV:hMPV co-formulation administered alone or in combination with a hPIV3 mixture was characterized. Mice were immunized with: a bivalent 1:1 co-formulation of hRSV:hMPV mRNA (each dose containing 0.5 µg of pre-fusion hRSV mRNA and 0.5 µg of pre-fusion hMPV mRNA); a bivalent 1:1 co-formulation of hRSV:hMPV mRNA administered with a hPIV3 mixture (containing 0.5 µg / dose of pre-fusion hPIV3 mRNA) (each dose containing 0.5 µg of pre-fusion hRSV mRNA and 0.5 µg of pre-fusion hMPV mRNA); or monovalent hPIV3 as a control (containing 0.5 µg / dose of pre-fusion hPIV3 mRNA). Each mRNA construct was prepared using LNP GL-HEPES-E3-E12-DS-4-E10 and diluted in PBS. Mice were boosted 21 days after primary immunization using the same protocol, and blood was collected before the first immunization (day 0), before the second immunization (day 21), and 2 weeks after the second immunization (day 35). The binding antibody titer was determined by ELISA using plates coated with pre-fusion hMPV, hPIV3, or hRSV. Figure 12A Neutralizing antibody titers were determined by micro-neutralization against hMPV, hPIV3, or hRSV expressing GFP reporter antibodies. Figure 12B The dashed line represents the lower limit of quantitation. In all assays, all day 0 samples were below the lower limit of quantitation. All samples were tested against all three targets. Error bars represent one geometric standard deviation from the geometric mean. Two-way ANOVA using a mixed model was employed. p < 0.05, p < 0.01, Statistical analysis was performed using p < 0.001. Detailed Implementation

[0153] This disclosure relates in particular to novel RNA (e.g., mRNA) compositions encoding combinations of hRSV F protein, hMPV protein, and / or hPIV3 protein, and methods for vaccination thereon. Specifically, this disclosure relates to mRNA encoding these proteins formulated in lipid nanoparticles (LNPs). I. Definition

[0154] Unless otherwise specified herein, scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials described herein may also be used in the practice or testing of this invention, exemplary methods and materials are described below. In case of conflict, this specification, including the definitions, shall prevail. Generally, the nomenclature and techniques used herein in conjunction with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and medicinal chemistry, protein and nucleic acid chemistry, and hybridization are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions as commonly practiced in the art or as described herein. Further, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout this specification and embodiments, the terms “have” and “comprise” or variations thereof (such as “has,” “having,” “comprises,” or “comprising”) should be understood to mean including the stated integers or groups of integers, but not excluding any other integers or groups of integers. All publications and other references cited herein are incorporated in their entirety by way of citation. While numerous documents are cited herein, this citation does not imply an admission that any of these documents constitutes part of common general knowledge in the art.

[0155] It should be noted that the terms “a” or “an” refer to one or more of the entities described; for example, “a nucleotide sequence” should be understood to represent one or more nucleotide sequences. Therefore, the terms “a or an”, “one or more”, and “at least one” are used interchangeably herein.

[0156] Furthermore, as used herein, “and / or” is considered a specific disclosure of whether each of the two specified features or components has or does not have the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used herein in phrases such as “A, B, and / or C” is intended to cover each of the following: 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 (alone); B (alone); and C (alone).

[0157] It should be understood that, regardless of the circumstances described herein with the language “including,” other similar aspects described with the phrasing “consisting of” and / or “substantially consisting of” are also provided.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press can provide a general dictionary for those skilled in the art of the matter for many of the terms used in this disclosure.

[0159] Units, prefixes, and symbols are all represented in their SI-acceptable form. Numerical ranges include numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right, from amino to carboxyl. The headings provided herein are not intended to limit the various aspects of this disclosure. Therefore, the terms defined below can be defined more fully by referring to the specification as a whole.

[0160] The term “about” or “approximately” is used herein to mean approximately, roughly, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the value. Generally, the term “about” can modify a value to be above or below (higher or lower) by variance (e.g., 10%, up or down). In some embodiments, the term indicates a deviation from the indicated value 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%. In some embodiments, “about” indicates a deviation from the indicated value of ±10%. In some embodiments, “about” indicates a deviation from the indicated value of ±5%. In some embodiments, “about” indicates a deviation from the indicated value of ±4%. In some embodiments, “about” indicates a deviation from the indicated value of ±3%. In some embodiments, “about” indicates a deviation from the indicated value of ±2%. In some embodiments, “about” indicates a deviation from the indicated value of ±1%. In some embodiments, "about" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.01% from the indicated value.

[0161] 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. mRNA may contain one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). Non-coding regions in mRNA include a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a 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.

[0162] As used herein, the term "antigenic site Ø" or "site Ø epitope" for RSV refers to the site located at the apex of the pre-fusion RSV F trimer containing amino acid residues 62-69 and 196-209 of wild-type RSV F (SEQ ID NO: 9). The RSV F site Ø epitope is a binding site for antibodies (such as D25 and AM14) that are specific to pre-fusion RSV F, and the binding of the antibody to the site Ø epitope blocks RSV cell surface attachment (see, for example, McLellan et al., Science 340(6136): 1113-1117, 2013). Recombinant human anti-RSV antibody D25 (Creative Biolabs®; catalog number: PABL-322) and recombinant human anti-RSV antibody AM14 (Creative Biolabs®; catalog number: PABL-321) are each commercially available.

[0163] As used herein, the term "antigenic site Ø" or "site Ø epitope" for hMPV refers to a site located in the pre-fusion form of the hMPV F trimer. The hMPV site Ø epitope is a binding site for antibodies specific to pre-fusion hMPV F.

[0164] As used herein, the term "antigenic site Ø" or "site Ø epitope" for hPIV3 refers to a site located in the pre-fusion form of the hPIV3 trimer. The hPIV3 site Ø epitope is a binding site for antibodies specific to pre-fusion hPIV3.

[0165] As used herein, the term "antigenic site V" or "site V epitope" refers to a site located in the pre-fusion form of the hMPV F trimer. Site V epitopes are binding sites for antibodies specific to pre-fusion hMPV F.

[0166] As used herein, the term "antigen stability" refers to the stability of an antigen over time or in solution.

[0167] As used herein, the term “cavity-filling substitution” refers to engineered hydrophobic substitution used to fill cavities present in pre-fusion RSV F trimer, pre-fusion hMPV F trimer, or pre-fusion hPIV3 trimer.

[0168] As used in this article, the term "RSV F protein" or "F protein" refers to the RSV protein responsible for driving the fusion of the viral envelope with the host cell membrane during viral entry.

[0169] As used in this article, the term "hMPV F protein" or "F protein" refers to the hMPV protein responsible for mediating the fusion of the viral envelope and the host cell membrane during viral entry.

[0170] As used in this article, the term "hPIV3 F protein" or "F protein" refers to the hPIV3 protein responsible for mediating the fusion of the viral envelope and the host cell membrane during viral entry.

[0171] As used herein, the term "RSV F polypeptide" refers to a polypeptide containing at least one epitope of the F protein.

[0172] As used herein, the term "hMPV F polypeptide" refers to a polypeptide containing at least one epitope of the hMPV F protein.

[0173] As used herein, the term "hPIV3 F polypeptide" refers to a polypeptide that contains at least one epitope of the hPIV3 F protein.

[0174] As used herein, the term "peptide" refers to any amino acid chain, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation). "Peptide" applies to amino acid polymers, including naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids (e.g., artificial chemical mimics of the corresponding naturally occurring amino acids). "Residue" refers to an amino acid or amino acid mimic incorporated into a peptide via an amide bond or an amide bond mimic. Peptides have an amino terminus (N-terminus) and a carboxyl terminus (C-terminus). "Peptide" is used interchangeably with peptide or protein and is used herein to refer to polymers containing amino acid residues.

[0175] As used herein, the term "glycan addition" refers to the addition of a mutation that introduces a glycosylation site absent in wild-type RSV F. This mutation can be engineered to increase construct expression, increase construct stability, or block epitopes shared between the pre-fusion and post-fusion conformations. Modified proteins including glycan addition will have more glycosylation and therefore a higher molecular weight. Glycan addition can reduce the extent to which RSV F peptides trigger antibodies against the post-fusion conformation of RSV F.

[0176] As used in this article, the "foldon domain" refers to the trimerized domain of T4 fibritin.

[0177] As used herein, a “signal peptide” or “signal sequence” refers to a peptide of approximately 16-30 amino acids in length that is located at the amino or carboxyl terminus of a polypeptide and whose function is to transfer the polypeptide from the endoplasmic reticulum and Golgi apparatus to the secretory pathway.

[0178] As used herein, a “tagged sequence” or “affinity tag” refers to a polypeptide sequence that can be used to purify a polypeptide or protein containing a tagged sequence. Tagged sequences include, for example, multihistidine tags (e.g., hexahistidine (6x His tag), octahistidine (8x His tag) etc.), glutathione S-transferase (GST), FLAG, streptavidin-binding peptide (SBP), strep II, maltose-binding protein (MBP), calmodulin-binding protein (CBP), chitin-binding domain (CBD), RNase A S protein, hemagglutinin (HA), c-Myc, etc.

[0179] As used herein, the term “intrapolymer stabilization substitution” refers to amino acid substitutions in RSV F, hMPV F, or hPIV3 F that stabilize the pre-fusion configuration through interactions within the protopolymer of RSV F trimer, hMPV F trimer, or hPIV3 trimer.

[0180] As used herein, the term “inter-protomer stabilization substitution” refers to amino acid substitutions in RSV F, hMPV F, or hPIV3 F that stabilize the pre-fusion configuration by stabilizing the interactions between the protomers of RSV F trimer, the protomers of hMPV F trimer, or the protomers of hPIV3 F trimer.

[0181] As used herein, the term "protease cleavage" refers to the proteolytic (sometimes also called "shearing") of susceptible residues (e.g., lysine or arginine) in a polypeptide sequence. Protease cleavage sites include viral protease cleavage sites, such as the hMPV F0 protease cleavage site, RSV F0 protease cleavage site, human rhinovirus 3C (HRV-3C) protease cleavage site, and hPIV3 F0 protease cleavage site.

[0182] As used in this article, the term "post-fusion" for RSV F, hMPV F, and hPIV3 F refers to the stable conformation of RSV F, hMPV F, and hPIV3 F that appears after the virus fuses with the cell membrane.

[0183] As used in this article, the term “pre-fusion” for RSV F, hMPV F, and hPIV3 F refers to the configuration of RSV F, hMPV F, and hPIV3 F used prior to virus-cell interaction.

[0184] As used herein, the term "protomer" refers to the structural unit of an oligomeric protein. In the case of RSV F, the individual unit of the RSV F trimer is a protomer. In the case of hMPVF, the individual unit of the hMPV F trimer is a protomer. In the case of hPIV3 F, the individual unit of the hPIV3 F trimer is a protomer.

[0185] As used herein, the term "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of the N (asparagine) residue. This is achieved through N-glycosylation, forming an N-glycan. This glycan can be a polysaccharide.

[0186] As used in this article, the term "glycosylation" refers to the addition of sugar units to a protein.

[0187] As used herein, the term "immune response" refers to the response of cells of the immune system (such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells) to a stimulus (such as an antigen or vaccine). An immune response can include any cell in the body involved in the 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.

[0188] As used herein, “protective immune response” refers to an immune response that protects a subject from infection (e.g., prevents infection or the occurrence of infection-related diseases). Methods for measuring immune responses include measuring, for example, the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc.

[0189] As used in this article, "antibody response" refers to the immune response that produces antibodies.

[0190] As used herein, “antigen” refers to a factor that elicits an immune response; and / or a factor that binds to T cell receptors (e.g., when presented by MHC molecules) or to antibodies (e.g., produced by B cells) when exposed to or administered to an organism. In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Alternatively or additionally, in some embodiments, an antigen elicits a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism species. In some embodiments, an antigen elicits 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 organism species. In some embodiments, the antigen binds to antibodies and / or T-cell receptors and may or may not induce a specific physiological response in an organism. In some embodiments, for example, the antigen may bind to antibodies and / or T-cell receptors in vitro, regardless of whether such interaction occurs in vivo. In some embodiments, the antigen reacts with products of a specific humoral or cellular immunity. The antigen includes RSV peptides, hMPV peptides, and hPIV3 peptides encoded by mRNA as described herein.

[0191] As used herein, “adjuvant” refers to a substance or carrier that enhances the immune response to an antigen. Adjuvants may include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions of antigen solutions emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Sometimes, killed mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) may also be used as adjuvants (e.g., see 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 may also include biomolecules such as Toll-like receptor (TLR) agonists and co-stimulatory molecules.

[0192] As used herein, "antigenic polypeptide" means a polypeptide containing all or part of the RSV amino acid sequence, the amino acid sequence being of sufficient length to make the molecule antigenic with respect to RSV; a polypeptide containing all or part of the hMPV amino acid sequence, the amino acid sequence being of sufficient length to make the molecule antigenic with respect to hMPV; or a polypeptide containing all or part of the hPIV3 amino acid sequence, the amino acid sequence being of sufficient length to make the molecule antigenic with respect to hPIV3.

[0193] As used herein, “subject” means any member of the animal kingdom. In some embodiments, “subject” means a human. In some embodiments, “subject” means a non-human animal. In some embodiments, a subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, a non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). 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, a adolescent, or an infant. In some embodiments, the terms “subject” or “patient” are used and are intended to be used interchangeably with “subject”. In some exemplary embodiments, the subject is a preterm newborn (e.g., less than 37 weeks gestation), a newborn (e.g., 0-27 days old), an infant or toddler (e.g., 28 days to 23 months old), a child (e.g., 2 to 11 years old), an adolescent (e.g., 12 to 17 years old), an adult (e.g., 18 to 50 years old or 18 to 64 years old), or an older adult (e.g., 65 years of age or older). In an exemplary embodiment, the subject is 18 to 50 years of age. In other exemplary embodiments, the subject is an older adult (e.g., an adult 60 years of age or older).

[0194] As used herein, the terms "vaccination" or "vaccinate" refer to the administration of a composition intended to, for example, produce an immune response to a pathogenic factor. Vaccination may be administered before, during, and / or after exposure to a pathogenic factor and / or the occurrence of one or more symptoms, and in some embodiments, shortly before, during, and / or after exposure to a pathogenic factor. In some embodiments, vaccination comprises multiple administrations of a vaccination composition at appropriate intervals.

[0195] This disclosure describes nucleic acid sequences (e.g., DNA and RNA sequences) and amino acid sequences that have a certain degree of identity with a given nucleic acid sequence or amino acid sequence (reference sequence), respectively.

[0196] Sequence identity between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. Sequence identity between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0197] The terms "identity %", "sameness %", or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the most preferred alignment between sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed across the entire length of the sequences to be compared. The comparison of two sequences is typically performed by comparing the sequences with respect to segments or "comparison windows" after the most preferred alignment to identify local regions of the respective sequences. The best alignment for comparison can be performed manually or by means of the local homology algorithm described in Smith and Waterman, 1981, AdsApp. Math. [Advances in Applied Mathematics] 2, 482; the local homology algorithm described in Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48, 443; the similarity search algorithm described in Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88, 2444; or by means of computer programs using said algorithms (e.g., GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

[0198] The identity percentage is obtained by determining the number of identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0199] 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, consecutive nucleotides). In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0200] Nucleic acid sequences or amino acid sequences having a specific degree of identity with a given nucleic acid sequence or amino acid sequence may possess at least one functional property of the given sequence, and in some cases, are functionally equivalent to the given sequence. In some embodiments, nucleic acid sequences or amino acid sequences having a specific degree of identity with a given nucleic acid sequence or amino acid sequence are functionally equivalent to the given sequence.

[0201] As used herein, the term “kit” refers to a packaged set of related components, such as one or more compounds or compositions, and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants. II. RNA

[0202] The vaccine disclosed herein may comprise at least two types of ribonucleic acid (RNA), each of which contains an ORF encoding a recombinant F protein antigenic polypeptide, such as RSV F protein antigen, hMPV F protein antigen, or hPIV3 protein antigen. In some embodiments, these RNAs are messenger RNAs (mRNAs) each containing an ORF encoding an RSV F protein antigen, hMPV F protein antigen, or hPIV3 protein antigen. In some embodiments, the RNA (e.g., mRNA) further comprises at least one 5' UTR, 3' UTR, poly(A) tail, and / or 5' cap. II.A. 5' Cap

[0203] The 5' cap on mRNA can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also known as "m7G" or "cap-0") contains a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0204] Typically, a 5' cap is added as follows: First, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphate groups; then, guanosine triphosphate (GTP) is added to the terminal phosphate groups via guanylate transferase, creating a 5'5'5 triphosphate bond; 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. Other cap structures are described in U.S. Publication Nos. US 2016 / 0032356 and US 2018 / 0125989, which are incorporated herein by reference.

[0205] The 5' capping of polynucleotides can be performed concurrently with in vitro transcription reactions using the following chemical RNA cap analogs, according to the manufacturer's protocol, to produce 5'-guanosine cap structures: 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 RNA can be performed post-transcriptionally using a vaccinia virus capping enzyme to generate a cap 0 structure: m7G(5')ppp(5')G. A cap 1 structure can be generated using both a vaccinia virus capping enzyme and a 2'-O methyltransferase to produce: m7G(5')ppp(5')G-2'-O-methyl. A cap 2 structure can be generated from the cap 1 structure, followed by 2'-O-methylation of the penultimate nucleotide at the 5' end using a 2'-O methyltransferase. A cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the penultimate nucleotide at the 5' end using a 2'-O methyltransferase.

[0206] In some embodiments, the mRNA disclosed herein 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.

[0207] In some embodiments, the mRNA disclosed herein comprises the following 5' cap: . II.B. Untranslated Region (UTR)

[0208] In some embodiments, the mRNA disclosed herein includes a 5' and / or a 3' untranslated region (UTR). In the mRNA, the 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0209] In some embodiments, the mRNA disclosed herein may include a 5' UTR containing one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5' UTR may have a length of about 10 to 5,000 nucleotides. In some embodiments, the 5' UTR may have a length of about 50 to 500 nucleotides. In some embodiments, the 5' UTR has a length of at least about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, or about 650 nucleotides. The lengths are approximately 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1,000 nucleotides, 1,500 nucleotides, 2,000 nucleotides, 2,500 nucleotides, 3,000 nucleotides, 3,500 nucleotides, 4,000 nucleotides, 4,500 nucleotides, or 5,000 nucleotides.

[0210] In some embodiments, the mRNA disclosed herein may include a 3' UTR comprising one or more of the following: a polyadenylation signal, a protein binding site affecting the stability of the mRNA's position in the cell, or one or more binding sites of the miRNA. In some embodiments, the 3' UTR may have a length of 50 to 5,000 nucleotides or longer. In some embodiments, the 3' UTR may have a length of 50 to 1,000 nucleotides or longer. In some embodiments, the 3' UTR has a length of at least about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, and about... The lengths are approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, approximately 900 nucleotides, approximately 950 nucleotides, approximately 1,000 nucleotides, approximately 1,500 nucleotides, approximately 2,000 nucleotides, approximately 2,500 nucleotides, approximately 3,000 nucleotides, approximately 3,500 nucleotides, approximately 4,000 nucleotides, approximately 4,500 nucleotides, or approximately 5,000 nucleotides.

[0211] In some embodiments, the mRNA disclosed herein may contain a 5' or 3' UTR derived from a gene different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0212] In some embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNA (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycling enzymes) to increase mRNA stability. For example, the 5' UTR sequence may include a portion of the CMV, i.e., the early 1 (IE1) gene or a fragment thereof, to improve mRNA nuclease resistance and / or improve mRNA half-life. Including a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3' end or untranslated region of the mRNA is also considered. Generally, these modifications improve mRNA stability and / or pharmacokinetic properties (e.g., half-life) relative to their unmodified counterparts and include modifications, for example, to improve mRNA resistance to in vivo nuclease digestion.

[0213] Exemplary 5' UTR includes a sequence derived from the CMV, i.e., the early 1 (IE1) gene (US Publications 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO: 18) (US Publication 2016 / 0151409, which is incorporated herein by reference).

[0214] 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) bundle. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In some embodiments, the 5' UTR derived from the 5' UTR of a TOP gene lacks the 5' TOP motif (oligopyrimidine bundle) (e.g., U.S. Publications 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0215] In some embodiments, 5' UTR is derived from the ribosomal protein large 32 (L32) gene (US Publication No. 2017 / 0029847, ibid.).

[0216] In some embodiments, the 5' UTR is derived from the 5' UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Publication No. 2016 / 0166710, ibid.).

[0217] In some embodiments, the 5' UTR is derived from the 5' UTR of the ATP5A1 gene (US Publication No. 2016 / 0166710, ibid.).

[0218] In some embodiments, the internal ribosome entry site (IRES) is used instead of the 5' UTR.

[0219] In some embodiments, the 5' UTR contains the nucleic acid sequence of GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO:7).

[0220] In some embodiments, the 3' UTR contains the nucleic acid sequence CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO:8).

[0221] The 5'UTR and 3'UTR are described in further detail in International Publication No. WO 2012 / 075040 (which is incorporated herein by reference). II.C. Polyadenylated tail

[0222] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to the adenosine nucleotide sequence at the 3' end of an mRNA molecule. The poly(A) tail can confer stability to mRNA and protect it from exonuclease degradation. The poly(A) tail can enhance translation. In some embodiments, the poly(A) tail is substantially homopolymeric. For example, a 100-adenosine nucleotide poly(A) tail can substantially have a length of 100 nucleotides. In some embodiments, the poly(A) tail can be interrupted by at least one nucleotide different from the adenosine nucleotide (e.g., a nucleotide that is not adenosine nucleotide). For example, a 100-adenosine nucleotide poly(A) tail can have a length exceeding 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide different from the adenosine nucleotide or a segment of nucleotides). In some embodiments, the poly(A) tail comprises the following sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 19).

[0223] As used herein, “poly(A) tail” typically refers to RNA. However, in the context of this disclosure, the term also refers to the corresponding sequence in a DNA molecule (e.g., “poly(T) sequence”).

[0224] The poly(A) tail may contain about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0225] 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 some embodiments, the poly(A) tail is obtained in vitro using conventional chemical synthesis methods without transcription from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of RNA (after in vitro transcription of RNA) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilized poly(A) polymerases, for example, using the methods and means described in WO 2016 / 174271.

[0226] Nucleic acids can contain poly(A) tails obtained by enzymatic polyadenylation, with most nucleic acid molecules containing about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0227] In some embodiments, the nucleic acid may include a poly(A) tail derived from the template DNA, and may additionally include at least one additional poly(A) tail generated by enzymatic polyadenylation, for example as described in International Publication No. WO 2016 / 091391.

[0228] In some embodiments, the nucleic acid contains at least one polyadenylation signal.

[0229] In various embodiments, the nucleic acid may contain at least one multiple (C) sequence.

[0230] As used herein, the term "multiple (C) sequence" is intended to be a cytosine nucleotide sequence of up to about 200 cytosine nucleotides. In some embodiments, the multiple (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 multiple (C) sequence comprises about 30 cytosine nucleotides. II.D. Chemical Modification

[0231] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may contain at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain 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 some embodiments, the disclosed mRNA can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such analogs or derivatives 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), dihydrouracil, 2-thio-uracil, 4-thio-uracil, 5-carboxyl-... Methylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, piracetamidine, β-D-mannosyl-piracetamidine, aminophosphate, thiophosphate, peptide nucleotide, methylphosphonate, 7-deazoguanosine, 5-methylcytosine, and inosine.

[0232] In some embodiments, the disclosed mRNA may contain at least one chemical modification, including but not limited to pseudouridine, N1-methylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0233] In some embodiments, the chemical modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0234] In some embodiments, the chemical modification includes N1-methylpseuuridine.

[0235] 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.

[0236] 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.

[0237] Preparation of such analogues is described, for example, in U.S. Patent 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. II.E. mRNA synthesis

[0238] The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, the mRNA disclosed herein can be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. [Developmental and Comparative Immunology] 25(2):152-159; Brunelle et al. (2013) MethodsEnzymol. [Enzymological Methods] 530:101-14. In short, IVT is typically performed using: a linear or circular DNA template containing a promoter, a library of ribonucleoside 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 inhibitors. The exact conditions may vary depending on the particular application. The presence of these reagents is generally undesirable in the final mRNA product and can be considered as impurities or contaminants that can be purified or removed to provide uncontaminated and / or homogeneous mRNA suitable for therapeutic use. While mRNA from an in vitro transcription reaction may be required in some embodiments, mRNA from other sources may be used in accordance with this disclosure, including wild-type mRNA produced by bacteria, fungi, plants and / or animals.

[0239] In some embodiments, at least one mRNA is composed of the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Clustered (A) tail.

[0240] In some embodiments, at least one mRNA is composed of the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 4; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Clustered (A) tail.

[0241] In some embodiments, at least one mRNA is composed of the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 5; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Clustered (A) tail.

[0242] In some embodiments, at least one mRNA is composed of the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Clustered (A) tail.

[0243] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an open reading frame (ORF) encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0244] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 4; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0245] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 5; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0246] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0247] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0248] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 4; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0249] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 5; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0250] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0251] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0252] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 4; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0253] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 5; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0254] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

[0255] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10, IM-001, OF-02, or cKK-E10.

[0256] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 4; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10, IM-001, OF-02, or cKK-E10.

[0257] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 5; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence having SEQ ID NO: 8; and (v) clustered (A) tails; The mRNA is formulated in lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10, IM-001, OF-02, or cKK-E10.

[0258] In some embodiments, the composition comprises at least two messenger mRNAs, wherein each of the at least two mRNAs comprises an ORF encoding a recombinant F protein antigenic polypeptide, and wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ; (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having the nucleic acid sequence of SEQ ID NO: 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10, IM-001, OF-02, or cKK-E10.

[0259] In some embodiments, the length of the poly(A) tail is about 10 to about 500 adenosine nucleotides. III. RSV F protein

[0260] RSV is an antisense single-stranded RNA virus belonging to the family Pneumoviridae. RSV can cause respiratory infections. RSV is an enveloped virus with a glycoprotein (G protein), a small hydrophobic protein (SH protein), and a fusion protein (F protein) on its surface.

[0261] The RSV F protein is responsible for the fusion of the virus and the host cell membrane and has at least three conformations (pre-fusion, intermediate, and post-fusion conformations). In its pre-fusion conformation (Pre-F), the F protein exists as a trimer in which the major antigenic site Ø is exposed. Site Ø serves as the primary target for neutralizing antibodies generated by RSV-infected subjects (see Coultas et al., Thorax. [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 sites Ø and V are no longer exposed. Pre-F transitions to a transient intermediate conformation, allowing the F protein to insert into the host cell membrane, leading to fusion of the virus and the host cell membrane. The final conformational transition results in a more stable and elongated protein form (Post-F). Sites II and IV of the F protein are present in both the Pre-F and Post-F conformations. Site I is also present in both the Pre-F and Post-F conformations, but antibodies targeting site I bind more tightly to the Post-F conformation than to the Pre-F conformation.

[0262] This article provides RNA (e.g., mRNA) encoding the antigenic RSV F polypeptide.

[0263] In one aspect, this disclosure provides a respiratory syncytial virus (RSV) vaccine comprising messenger RNA (mRNA) containing 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 with SEQ ID NO: 1 or is composed of the amino acid sequence of SEQ ID NO: 1.

[0264] In some embodiments, epitopes of the RSV F protein shared between Pre-F and Post-F are blocked. When RNA (e.g., mRNA) encoding an antigenic RSV F polypeptide is administered to a subject, blocking the epitope reduces or eliminates antibody production against the epitope. This can increase the proportion of antibodies targeting epitopes specific to a particular conformation of F (e.g., the pre-fusion conformation), such as antibodies targeting site Ø. Because F has a pre-fusion conformation in the virus before it enters the cell, an increased proportion of antibodies targeting Pre-F can provide greater neutralization (e.g., expressed as a neutralization to binding ratio, as described herein). Blocking can be achieved by engineering bulk portions (e.g., N-glycans) near the shared epitope. For example, an N-glycosylation site not present in wild-type F can be added, for example, by mutating appropriate residues 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 the blocked epitope are also blocked, optionally wherein the blocked epitope is an epitope of antigenic site I of RSV F.

[0265] In some embodiments, the RSV F peptide contains asparagine substitution (i.e., E328N, S348N, or R507N) at one or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO: 9. In some embodiments, the RSV F peptide contains asparagine substitution (i.e., E328N, S348N, or R507N) at two or more positions corresponding to positions 328, 348, or 507 of SEQ ID NO: 9. In some embodiments, the RSV F peptide contains asparagine substitution (i.e., E328N, S348N, and R507N) at positions 328, 348, and 507 of SEQ ID NO: 9.

[0266] As previously indicated, such asparagines have been found to act as glycosylation sites (see International Publication No. WO2019 / 195291, incorporated herein by reference). Furthermore, without being bound by any particular theory, when RNA (e.g., mRNA) encoding antigenic RSV F peptides is administered to a subject, the glycans at these sites can inhibit antibody production against nearby epitopes, including epitopes shared by pre- and post-fusion RSV F proteins. In some embodiments, glycosylation of asparagine at positions 328, 348, or 507 of SEQ ID NO: 9 blocks at least one epitope shared between pre- and post-fusion RSV F, such as the epitope of antigenic site 1. Inhibiting antibody production against epitopes shared by pre- and post-fusion RSV F proteins may be beneficial because it can guide antibody production against epitopes specific to the pre-fusion RSV F protein (such as site Ø epitope), which may have more potent neutralizing activity than antibodies against other RSV F epitopes. The epitope relates to amino acid residues 62-69 and 196-209 of SEQ ID NO: 9. Therefore, in some embodiments, the RSV F polypeptide comprises amino acid residues 62-69 and 196-209 of SEQ ID NO: 9.

[0267] The RSV F peptides described herein may have deletions or substitutions of varying lengths relative to wild-type RSV F. For example, in the RSV F peptide of SEQ ID NO: 9, positions 98-144 of the wild-type sequence (SEQ ID NO: 9) are substituted with GSGNVGL (SEQ ID NO: 20), resulting in a net removal of 40 amino acids, such that positions 328, 348, or 507 of SEQ ID NO: 9 correspond to positions 288, 308, and 467 of SEQ ID NO: 1. In an alternative embodiment, in the RSV F peptide of SEQ ID NO: 1, positions 98-146 of the wild-type sequence (SEQ ID NO: 9) are substituted with GSGNVGLGG (SEQ ID NO: 21, positions 98-106 of SEQ ID NO: 1), resulting in a net removal of 40 amino acids, such that positions 328, 348, or 507 of SEQ ID NO: 9 correspond to positions 290, 310, and 469 of SEQ ID NO: 1.

[0268] Generally, positions in the constructs described herein can be mapped to the wild-type sequence of SEQ ID NO: 9 using pairwise alignment, for example, by the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, vacancy penalty of 10, vacancy extension penalty of 0.5). See also the discussion of structural alignments, as provided herein, as alternative methods for identifying the corresponding positions.

[0269] In some embodiments, the RSV F peptide contains a mutation that adds a glycan to block epitopes on the pre-fusion antigen that are structurally similar to those epitopes on the surface of the post-fusion RSV F. In some embodiments, the glycan is added to specifically block epitopes that may be present in the post-fusion conformation of RSV F. In some embodiments, a glycan is added that blocks epitopes that may be present in the post-fusion conformation of RSV F but does not affect one or more epitopes (such as site Ø epitopes) present in the pre-fusion conformation of RSV F.

[0270] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity with the amino acid sequences shown below: (SEQ ID NO: 22)

[0271] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity with the amino acid sequences shown below: (SEQ ID NO: 1)

[0272] In some embodiments, the RSV F peptide comprises DS-CAV1 amino acid substitutions (as described, for example, in McLellan et al., Science, 342(6158): 592-598, 2013), wherein further modifications have been made, including at least one, two, or three of the asparagines described above. The CAV1 mutations relative to SEQ ID NO: 9 are S190F and V207L. The DS mutations relative to SEQ ID NO: 9 are S155C and S290C.

[0273] In some embodiments, the amino acid substitution or amino acid substitution pair is an inter-protopolymer stabilizing substitution. Exemplary substitutions that may be inter-protopolymer stabilizing substitutions are V207L; N228F; I217V and E218F; I221L and E222M; or Q224A and Q225L (using position numbers of SEQ ID NO: 9).

[0274] In some embodiments, the amino acid substitution or amino acid substitution pair is stabilized within the protopolymer. Exemplary substitutions that may be stabilized within the protopolymer are V220I; and A74L and Q81L (using position numbers of SEQ ID NO: 9).

[0275] In some embodiments, the amino acid substitution is helically stabilized, i.e., it is predicted to stabilize the helical domain of RSV F. Stabilization of the helical domain can generally contribute to the stability of the Ø epitope and the pre-fusion conformation of RSV F. Exemplary substitutions that can be helically stabilized are N216P or I217P (using position numbers in SEQ ID NO: 9). Position 217 in SEQ ID NO: 9 corresponds to position 177 in SEQ ID NO: 1.

[0276] In some embodiments, the amino acid substitution is helical capping. In some embodiments, the amino acid substitution is helical PRO capping. Helical capping is based on the following biophysical observation: while a mutational placement of proline residues in the α-helix may disrupt helix formation, the proline at the N-terminus of the helical region can help induce helix formation by stabilizing the PHI / PSI bond angle. Exemplary substitutions that can be helical capping are N216P or I217P (using position numbers from SEQ ID NO: 9).

[0277] In some embodiments, amino acid substitutions replace disulfide mutations in DS-CAV1. In some embodiments, engineered disulfides of DS-CAV1 are reverted to the wild-type of DS-CAV1 (C69S and / or C212S mutations) (using position numbers from SEQ ID NO: 9). In some embodiments, one or more C residues of DS-CAV1 are substituted with S residues to eliminate disulfide bonds. In some embodiments, C69S or C212S substitution using position numbers from SEQ ID NO: 9 eliminates disulfide bonds. In some embodiments, the RSV F polypeptide comprises both C69S and C212S using position numbers from SEQ ID NO: 9. In some embodiments, substitution of such cysteine ​​residues and thereby elimination of disulfide bonds prevents the reduction of the RSV F polypeptide (i.e., acceptance of electrons from a reducing agent). In some embodiments, the antigen comprises I217P substitution using position numbers from SEQ ID NO: 9 instead of substitutions at C69 and / or C212.

[0278] In some embodiments, amino acid substitution prevents proteolysis by trypsin or trypsin-like proteases. In some embodiments, amino acids that prevent such proteolysis are substituted in the heptapeptide repeat region B (HRB) of RSV F.

[0279] The presence of fragments consistent with the proteolysis of RSV F peptides containing the wild-type HRB region indicates that lysine or arginine in this region is a target of proteolysis. Amino acid substitutions used to remove K or R residues can be termed knockout (KO). In some embodiments, K or R substitutes for L or Q. In some embodiments, K substitutes for L or Q. In some embodiments, the RSV F peptide comprises K498L and / or K508Q using position numbers from SEQ ID NO: 9. The corresponding positions in SEQ ID NO: 1 are 458 and 468, respectively. In some embodiments, the RSV F peptide comprises both K498L and K508Q.

[0280] In some embodiments, amino acid substitution is used to add a glycan. In some embodiments, amino acid substitution increases glycosylation by adding a glycan to the RSV F peptide. Substitution for adding a glycan can also be referred to as engineered glycosylation, compared to natural glycosylation (without additional glycans).

[0281] In some embodiments, the amino acid substitution used to add the glycan is N-substituted. In some embodiments, the N-substituted amino acid allows for N-linked glycosylation. In some embodiments, N substitution is accompanied by T or S substitution at the second amino acid position at the C-terminus of N, thereby forming an NxT / S glycosylation motif. In some embodiments, N is surface-exposed.

[0282] Each of the substitutions and mutations listed above in the RSV F polypeptide is described in more detail in International Publication No. WO 2019 / 195291, which is incorporated herein by reference.

[0283] In one aspect, this disclosure provides an RSV vaccine comprising messenger RNA (mRNA) containing an open reading frame (ORF) encoding an RSV F protein antigen, wherein the RSV F protein antigen comprises one or more of the following substitutions relative to the amino acid sequence shown in SEQ ID NO: 9:

[0284] 1) Amino acid positions 98-146 of SEQ ID NO: 9 are replaced by the amino acid sequence GSGNVGLGG (SEQ ID NO: 21);

[0285] 2) Amino acid substitutions for S190F and V207L;

[0286] 3) Amino acid substitution of I217P;

[0287] 4) Amino acid substitutions for E328N, S348N, and R507N;

[0288] 5) Amino acid substitution for L373R;

[0289] 6) Amino acid substitution of K498L; and

[0290] 7) Amino acid substitution for K508Q.

[0291] On the other hand, this disclosure provides an RSV vaccine comprising mRNA containing an ORF encoding an RSV F protein antigen, wherein, relative to the amino acid sequence shown in SEQ ID NO: 9, the RSV F protein antigen comprises each of the following substitutions:

[0292] 1) Amino acid positions 98-146 of SEQ ID NO: 9 are replaced by the amino acid sequence GSGNVGLGG (SEQ ID NO: 21);

[0293] 2) Amino acid substitutions for S190F and V207L;

[0294] 3) Amino acid substitution of I217P;

[0295] 4) Amino acid substitutions for E328N, S348N, and R507N;

[0296] 5) Amino acid substitution for L373R;

[0297] 6) Amino acid substitution of K498L; and

[0298] 7) Amino acid substitution for K508Q.

[0299] In some embodiments, the RSV F protein antigen comprises a transmembrane domain and a cytoplasmic tail amino acid sequence IMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 23).

[0300] 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 with the nucleic acid sequence shown in any one of the following SEQ ID NOs: 24-26.

[0301] 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 with the nucleic acid sequence shown in any one of the following SEQ ID NOs: 27-29

[0302] In some embodiments, the ORF (e.g., the ORF of hRSV, hMPV, or hPIV3) is codon-optimized. As used herein, "codon-optimized" or "codon-enhanced" means the introduction of certain codons (to exchange corresponding wild-type codons encoding the same amino acid), which may be more beneficial to RNA stability and / or the codon usage of the subject.

[0303] In some embodiments, the hRSV F protein antigen is encoded by a codon-optimized mRNA ORF as shown below: IV.hMPV F protein

[0304] hMPV is a negative-sense, single-stranded RNA virus belonging to the subfamily Pneumovirinae of the family Paramyxoviridae. hMPV infects airway epithelial cells in the nose and lungs and is the second most common cause of lower respiratory tract infections in young children, after respiratory syncytial virus (RSV). hMPV is an enveloped virus with glycoproteins (G protein), small hydrophobic proteins (SH protein), and fusion proteins (F protein) on its virion surface.

[0305] Because hMPV is an enveloped virus, its entry into host cells requires viral-cell membrane fusion. Paramyxovirus entry typically requires two viral glycoproteins, a fusion (F) protein and an attachment (G, H, or HN) protein, and all detected paramyxovirus glycoprotein-promoted membrane fusions occur at neutral pH, with one possible exception (i.e., SER virus). In addition to virus-cell membrane fusion, paramyxovirus glycoproteins also promote cell-cell fusion. Multinucleated giant cells (called syncytia) can be found in tissues infected with various paramyxoviruses. Cultured cells infected with hMPV form syncytia, but examination of primary human airway epithelial cells infected with hMPV suggests that syncytia formation may not occur frequently in vivo.

[0306] hMPV F is a class I fusion glycoprotein synthesized from an inactive precursor (F0) that requires cleavage to acquire fusion capability. Proteolytic cleavage produces two disulfide-linked subunits (F2 located at the N-terminus of F1), which assemble into a homotrimer. Cleavage occurs at a single-basic cleavage site immediately upstream of the hydrophobic fusion peptide. Cleavage can be achieved in tissue culture by adding exogenous trypsin to the culture medium or by adding a furin expression plasmid. However, in vivo, other serine proteases (such as TMPRSS2) are considered to be more relevant to cleavage. The F trimer is incorporated into the viral particle in a metastable "pre-fusion" or "pre-F" conformation. To initiate membrane fusion, hMPV F is activated and undergoes a series of progressive conformational changes in the F protein that drive membrane fusion and result in hMPV F adopting a highly stable "post-fusion" or "post-F" conformation.

[0307] In some exemplary embodiments, F0 proteolytic cleavage is achieved by co-transfection of a plasmid encoding the hMPV F polypeptide and a plasmid encoding furin (at a ratio of 4:1 hMPV plasmid: furin plasmid).

[0308] This document provides antigenic hMPV peptides comprising the hMPV F peptide. The hMPV F peptide may comprise the full sequence of hMPV F or a portion of hMPV F. In some embodiments, the portion is an extracellular domain.

[0309] In some embodiments, the hMPV F polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any one of SEQ ID NO: 10, 31, 33, and 35.

[0310] In some embodiments, the hMPV F polypeptide comprises a modified hMPV F polypeptide having at least 80% identity with a polypeptide of any one of SEQ ID NO: 10, 31, 33 and 35, wherein the hMPV F polypeptide is antigenic.

[0311] In some embodiments, the hMPV F polypeptide contains only the extracellular domain portion of the F protein.

[0312] The amino acid sequence of F0 in A2-CAN97-83 is: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGV AIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLM LENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTG RHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNT GFIIVIILIAVLGSSMILVSIFII IKKTKKPTGAPPELSGVTNNGFIPHN (SEQ ID NO: 10). (Accession number AAN52910; version AAN52910.1; DB source accession number AY145296.1.) The transmembrane domain is bolded and underlined, and the cytoplasmic tail is thickened.

[0313] The nucleotide sequence of F0 in A2-CAN97-83 is:

[0314] In some embodiments, epitopes of the hMPV F protein shared between pre-F and post-F are blocked. Blocking the epitope reduces or eliminates antibody production against the epitope when the RNA (e.g., mRNA) encoding the antigenic hMPV F peptide is administered to a subject. This can increase the proportion of antibodies targeting epitopes specific to a particular conformation of F (e.g., the pre-fusion conformation), such as antibodies targeting site Ø and / or site V. Because F has a pre-fusion conformation in the virus before it enters the cell, the increased proportion of antibodies targeting pre-F can provide greater neutralization (e.g., expressed as a neutralization to binding ratio, as described herein).

[0315] The hMPV F peptide described herein may have deletions or substitutions relative to the wild-type hMPV F protein (e.g., SEQ ID NO:10).

[0316] For example, in some embodiments, the hMPV polypeptide: (a) lacks a transmembrane domain and a cytoplasmic tail, and contains a human rhinovirus 3C (HRV-3C) protease cleavage site; (b) contains an F0 cleavage site mutation relative to SEQ ID NO: 10, the mutation comprising amino acid substitutions Q100R and S101R, i.e., glutamine at amino acid position 100 is replaced with arginine, and serine at amino acid position 101 is replaced with arginine; (c) contains a heterologous signal peptide; (d) contains at least one tag sequence (optionally a multihistidine tag, e.g., a 6x His tag, an 8x His tag, etc.) and / or a Strep II tag; and / or (e) contains a folded domain.

[0317] In some embodiments, the hMPV peptide lacks a transmembrane domain and a cytoplasmic tail, and comprises: an F0 cleavage site mutation relative to SEQ ID NO: 10, the mutation comprising amino acid substitutions Q100R and S101R; namely, glutamine at amino acid position 100 replaced by arginine, and serine at amino acid position 101 replaced by arginine; a human rhinovirus 3C (HRV-3C) protease cleavage site; a heterologous signal peptide; multiple histidine tags (e.g., 6x His tags, 8x His tags, etc.) and / or Strep II tags; and a folded domain.

[0318] In some embodiments, the hMPV peptide includes a valine, alanine, glycine, isoleucine, leucine, or proline substitution at position 185 of SEQ ID NO: 10.

[0319] In some embodiments, the hMPV polypeptide includes a phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine substituted at position 160 of SEQ ID NO: 10, and / or a valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline substituted at position 46 of SEQ ID NO: 10.

[0320] In some embodiments, the hMPV peptide includes a substitution at position 160 of SEQ ID NO: 10 and a substitution at position 46 of SEQ ID NO: 10, wherein these substitutions are “stabilizing substitutions” that stabilize the tertiary and / or quaternary structures of the hMPV peptide. Stabilizing substitutions include, but are not limited to, substitutions of the following: hydrophobic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, and methionine); hydrophilic amino acids (e.g., cysteine, serine, threonine, asparagine, and glutamine); disulfide-forming amino acids (e.g., cysteine); hydrogen-forming amino acids (e.g., tryptophan, histidine, tyrosine, and phenylalanine); charged amino acids (e.g., aspartic acid, glutamic acid, arginine, lysine, and histidine), etc.

[0321] In some embodiments, the hMPV peptide is derived from strain A hMPV (e.g., subtype A1 or A2) or from strain B hMPV (e.g., subtype B1 or B2).

[0322] In some embodiments, an amino acid sequence comprising the "backbone" F0 polypeptide sequence is provided, as shown below: (SEQ ID NO: 31). (Lowercase amino acids represent linker, fold motif, linker, HRV-3C cleavage site, linker, 8X-His tag, and strep tag II region.)

[0323] In some embodiments, a nucleotide sequence encoding the F0 polypeptide sequence is provided, as shown below:

[0324] In some embodiments, the hMPV polypeptide comprises the "backbone" hMPV sequence as shown in SEQ ID NO: 31, and may optionally contain one or more amino acid substitutions. For example, in some embodiments, the hMPV polypeptide includes a valine, alanine, glycine, isoleucine, leucine, or proline substitution at position 185 of SEQ ID NO: 31. In some embodiments, the hMPV polypeptide includes a phenylalanine, tryptophan, or tyrosine substitution at position 160 of SEQ ID NO: 31, and / or a valine, alanine, glycine, isoleucine, leucine, or proline substitution at position 46 of SEQ ID NO: 31. In some embodiments, the hMPV polypeptide includes an arginine substitution at one or both of positions 100 and 101 of SEQ ID NO: 31.

[0325] In some embodiments, the hMPV peptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 31.

[0326] In some embodiments, an amino acid sequence comprising the hMPV polypeptide sequence is provided, as shown below: (SEQ ID NO: 33) (D185P). (Lowercase amino acids represent linker, fold motif, linker, HRV-3C cleavage site, linker, 8X-His tag, and strep tag II region.)

[0327] In some embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided, as shown below:

[0328] In some embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided, as shown below:

[0329] In some embodiments, the hMPV polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 33. In some embodiments, the hMPV polypeptide comprises SEQ ID NO: 33. In some embodiments, the hMPV polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 34. In some embodiments, the hMPV polynucleotide comprises SEQ ID NO: 34. In some embodiments, the hMPV polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 36. In some embodiments, the hMPV polynucleotide comprises SEQ ID NO: 36.

[0330] In some embodiments, an amino acid sequence comprising the hMPV polypeptide sequence is provided, as shown below: (SEQ ID NO: 35) (T160F_N46V). (Lowercase amino acids represent linker, fold motif, linker, HRV-3C cleavage site, linker, 8X-His tag, and strep tag II region.)

[0331] In some embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided, as shown below:

[0332] In some embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided, as shown below:

[0333] In some embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided, as shown below:

[0334] In some embodiments, the hMPV polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 35. In some embodiments, the hMPV polypeptide comprises SEQ ID NO: 35. In some embodiments, the hMPV polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 37. In some embodiments, the hMPV polynucleotide comprises SEQ ID NO: 37. In some embodiments, the hMPV polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 38. In some embodiments, the hMPV polynucleotide comprises SEQ ID NO: 38. In some embodiments, the hMPV polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 37. In some embodiments, the hMPV polynucleotide comprises SEQ ID NO: 37. In some embodiments, the hMPV polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 39. In some embodiments, the hMPV polynucleotide comprises SEQ ID NO: 39.

[0335] Generally speaking, the positions in the constructs described in this paper can be mapped to reference sequences (e.g., the wild-type sequence of SEQ ID NO: 10 or the skeleton sequence of SEQ ID NO: 31) by pairwise alignment, for example, using the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, vacancy penalty of 10, vacancy extension penalty of 0.5).

[0336] In some embodiments, the vaccine disclosed herein may comprise at least one hMPV F protein antigen. The hMPV F protein antigen disclosed herein can be prepared by a variety of methods. In one embodiment, a host cell line, possibly of eukaryotic or prokaryotic origin, is used to express the hMPV F polypeptide. In one embodiment, the host cell line used to express the hMPV F polypeptide is bacterial. In one embodiment, the host cell line used to express the hMPV F polypeptide is mammalian. Specific host cell lines best suited for expressing the desired gene product can be determined. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical cancer), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), CHO (Chinese hamster ovary), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). Host cell lines are typically available from commercial services, the American Tissue Culture Collection (ATCC), or from publicly available literature.

[0337] In some embodiments, the hMPV F protein antigen is shown as: (SEQ ID NO: 40) (A2-D185P).

[0338] In some embodiments, the hMPV F protein antigen is encoded by an mRNA ORF (A2-D185P mRNA ORF) as shown in (SEQ ID NO: 34).

[0339] In some embodiments, the hMPV F protein antigen is encoded by a codon-optimized mRNA ORF (AD185P mRNA ORF) as shown in (SEQ ID NO: 36).

[0340] In some embodiments, the hMPV F protein antigen is shown as: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTvVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLAfAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN (SEQ ID NO: 2) (A2-T160F_N46V).

[0341] In certain embodiments, the hMPV F protein antigen is encoded by the following codon-optimized mRNA ORF: (SEQ ID NO:5)

[0342] In some embodiments, the hMPV F protein antigen is encoded by an mRNA ORF (A2-T160F_N46V mRNA ORF) as shown in (SEQ ID NO: 37).

[0343] In some embodiments, the hMPV F protein antigen is encoded by a codon-optimized mRNA ORF (T160F_N46V mRNA ORF) as shown in (SEQ ID NO: 38).

[0344] In some embodiments, the hMPV F protein antigen is encoded by a codon-optimized mRNA ORF (T160F_N46V mRNA ORF) as shown in (SEQ ID NO: 39). V.hPIV3 F protein

[0345] hPIV3 is an enveloped, unsegmented, negative-sense, single-stranded RNA virus belonging to the subfamily Paramyxovirinae of the family Paramyxoviridae. It is a major cause of childhood illness and hospitalization worldwide. Entry of hPIV3 requires viral-cell membrane fusion, catalyzed by the hPIV3 fusion (F) protein on the virion surface.

[0346] hPIV3 F is a class I fusion glycoprotein expressed as an inactive precursor (F0), which requires cleavage to acquire fusion capability. During Golgi apparatus transport, F0 undergoes proteolytic cleavage, producing disulfide-linked heterodimers of F2 and F1 polypeptides (F2 at the N-terminus of F1). The three protomers of the F2-F1 heterodimer assemble to form the mature F protein, which adopts a metastable "pre-fusion" or "pre-F" conformation. To initiate membrane fusion, hPIV3 F is activated and undergoes a series of progressive conformational changes within the F protein. These changes drive membrane fusion and result in hPIV3 F adopting a highly stable "post-fusion" or "post-F" conformation.

[0347] This document provides antigenic hPIV3 peptides comprising the hPIV3 F peptide. The hPIV3 F peptide may comprise the full sequence of hPIV F or a portion of hPIV F. In some embodiments, the portion is an extracellular domain.

[0348] The hPIV3 F peptide described herein can have deletions or substitutions relative to the wild-type hPIV3 F protein shown below: MLISILSIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVAL VEAKQAKSDIEKLKEAIRDTNKAVQSVQSSVGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDV DLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTTCTCNGIGNRINQP PDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGSWHQSSTTIIIILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK (SEQ ID NO: 11).

[0349] For example, in some embodiments, the hPIV3 F polypeptide comprises amino acid substitutions of Q162C, L168C, I213C, G230C, A463V, and I474Y relative to SEQ ID NO: 11.

[0350] In some embodiments, the hPIV3 F extracellular domain trimer includes a protopolymer comprising one or more amino acid substitutions or deletions that stabilize the hPIV3 extracellular domain trimer in a pre-fusion configuration, wherein the one or more amino acid substitutions or deletions include one or more of the following group of substitutions for forming disulfide bonds to stabilize the hPIV3 extracellular domain trimer in a pre-fusion configuration: 162C and 168C; 213C and 230C.

[0351] In some embodiments, the promerogenum of the hPIV3 F extracellular domain trimer comprises, at positions 19-481 of hPIV3 F, amino acid substitutions (e.g., K108E) for removing the shared furin cleavage site between F2 and F1 (if the shared site is present in the native sequence), a non-native disulfide bond substitution between 162C and 168C (e.g., Q162C and L168C), and a linking to the C-terminal GCN4 trimerizing domain (e.g., via an SA peptide linker). In some such embodiments, the promerogenum further comprises cavity-filling substitutions of 463V and 474Y (e.g., A463V and I474Y) for stabilization of the pre-fusion conformation.

[0352] In some embodiments, the promerum of the hPIV3 F extracellular domain trimer comprises, at positions 19-481 of hPIV3 F, amino acid substitutions (e.g., K108E) for removing the shared furin cleavage site between F2 and F1 (if the shared site is present in the native sequence), substitutions for non-natural disulfide bonds between 162C and 168C (e.g., Q162C and L168C), substitutions for non-natural disulfide bonds between 213C and 230C (e.g., 1213C and G230C), and a linking to the C-terminal GCN4 trimerizing domain (e.g., via an SA peptide linker). In some such embodiments, the promerum further comprises cavity-filling substitutions of 463V and 474Y (e.g., A463V and I474Y) for stabilization of the pre-fusion conformation.

[0353] In some embodiments, the promeron of the hPIV3 F extracellular domain trimer comprises a full-length hPIV3 F protein (minus the signal peptide) containing: an amino acid substitution (e.g., K108E) for removing the shared furin cleavage site between F2 and F1 (if the shared site is present in the native sequence) and a non-native disulfide bond substitution (e.g., Q162C and L168C) between 162C and 168C. ​​In some such embodiments, the promeron further comprises 463V and 474Y (e.g., A463V and I474Y) cavity-filling substitutions for stabilization of the pre-fusion conformation.

[0354] In some embodiments, the promerum of the hPIV3 F extracellular domain trimer comprises the full-length hPIV3 F protein (minus the signal peptide) containing: amino acid substitutions (e.g., K108E) for removing the shared furin cleavage site between F2 and F1 (if the shared site is present in the native sequence), non-native disulfide bond substitutions (e.g., Q162C and L168C) between 162C and 168C, and non-native disulfide bond substitutions (e.g., I213C and G230C) between 213C and 230C. In some such embodiments, the promerum further comprises cavity-filling substitutions of 463V and 474Y (e.g., A463V and I474Y) for stabilization of the pre-fusion conformation.

[0355] In some embodiments, an amino acid sequence comprising the hPIV3 F polypeptide sequence is provided, as shown below: MLISILSIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVAL VEAKQAKSDIEKLKEAIRDTNKAVQSVCSSVGNCIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNCFGDNIGSLQEKGIKLQCIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDV DLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTTCTCNGIGNRINQP PDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKVKSDLEESKEWYRRSNQKLDSIGSWHQSSTTIIIILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK (SEQ ID NO:3)

[0356] In some embodiments, a nucleotide sequence encoding a codon-optimized hPIV3 F polypeptide sequence is provided, as shown below: (SEQ ID NO: 14)

[0357] In some embodiments, the hPIV3 F polypeptide comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.5% identity with any one of SEQ ID NO: 3 and 11.

[0358] In some embodiments, the hPIV3 F polypeptide comprises a modified hPIV3 F polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.5% identity with a polypeptide of any one of SEQ ID NO: 3 and 11, wherein the hPIV3 F polypeptide is antigenic.

[0359] In some embodiments, the hPIV3 F polypeptide contains only the extracellular domain portion of the F protein.

[0360] This article provides the RNA (e.g., mRNA) encoding the antigenic hPIV3 F peptide.

[0361] In one aspect, this disclosure provides an hPIV3 vaccine comprising mRNA containing an open reading frame (ORF) encoding an hPIV3 F protein antigen, wherein the hPIV3 F protein antigen comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.5% identity with or consisting of the amino acid sequence of SEQ ID NO: 3.

[0362] Generally, positions in the constructs described herein can be mapped to the wild-type sequence of SEQ ID NO: 11 using pairwise alignment, for example, by the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, vacancy penalty of 10, vacancy extension penalty of 0.5). See also the discussion of structural alignments, as provided herein, as alternative methods for identifying the corresponding positions.

[0363] In some embodiments, the hPIV3 F protein antigen is encoded by a codon-optimized mRNA ORF as shown below: (SEQ ID NO:6)

[0364] The recombinant hPIV3 F peptide is described in further detail in U.S. Patent No. 11,078,239, which is incorporated herein by reference. VI. Combined respiratory mRNA vaccine composition

[0365] This article provides compositions that target two or more of the three most common respiratory pathogens associated with high morbidity and mortality in infants, children, and the elderly: hRSV, hMPV, and hPIV3. The combined disease burden of these respiratory pathogens exceeds that of influenza. Because there are currently no available vaccines or specific therapies, there is an unmet need for safe, effective, and convenient mRNA combination vaccines to protect individuals from these pathogens.

[0366] In some embodiments, the composition comprises at least two mRNAs, wherein the at least two mRNAs comprise an open reading frame (ORF) encoding a recombinant F protein antigenic polypeptide, selected from the group consisting of: (i) a first mRNA encoding an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; (ii) a second mRNA encoding an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a third mRNA encoding an hPIV3F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0367] In some embodiments, the above composition comprises at least two mRNAs, wherein the at least two mRNAs comprise an ORF encoding a recombinant F protein antigenic polypeptide, including: (i) a first mRNA encoding an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a second mRNA encoding an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2.

[0368] In some embodiments, the composition comprises at least two mRNAs, wherein the at least two mRNAs comprise an ORF encoding a recombinant F protein antigenic polypeptide, including: (i) a first mRNA encoding an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a second mRNA encoding an hPIV3 F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0369] In some embodiments, the composition comprises at least two mRNAs, wherein the at least two mRNAs comprise an ORF encoding a recombinant F protein antigenic polypeptide, including: (i) a first mRNA encoding an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (ii) a second mRNA encoding an hPIV3 F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0370] In some embodiments, the composition comprises at least three mRNAs, wherein the at least three mRNAs comprise an ORF encoding a recombinant F protein antigenic polypeptide, including: (i) a first mRNA encoding an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a second mRNA encoding an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (ii) a second mRNA encoding an hPIV3 F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0371] In some embodiments, the composition comprises three mRNAs, wherein the three mRNAs comprise an ORF encoding a recombinant F protein antigenic polypeptide, including: (i) a first mRNA encoding an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a second mRNA encoding an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (ii) a second mRNA encoding an hPIV3 F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0372] In some embodiments, the composition comprises three mRNAs, wherein: (i) a first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; (ii) a second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a third mRNA encodes an hPIV3F protein antigen comprising the amino acid sequence of SEQ ID NO: 3.

[0373] In some embodiments, the composition comprises three mRNAs, wherein: (i) a first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; (ii) a second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a third mRNA encodes an hPIV3F protein antigen comprising the amino acid sequence of SEQ ID NO: 3, wherein the first mRNA, the second mRNA, and the third mRNA are formulated into the same LNP comprising: 40% of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, or IM-001; 1.5% of DMG-PEG2000; 28.5% of cholesterol; and 30% of DOPE.

[0374] In some embodiments, the composition comprises a first mRNA and a second mRNA, wherein: (i) the first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2, wherein the first mRNA and the second mRNA are formulated into the same LNP, the same LNP comprising: OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, or IM-001. This composition may be administered together with a separate composition comprising the hPIV3 F protein antigen. The hPIV3 F protein antigen may comprise the amino acid sequence of SEQ ID NO: 3.

[0375] In some embodiments, the composition comprises a first mRNA and a second mRNA, wherein: (i) the first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2, wherein the first mRNA and the second mRNA are formulated into separate LNPs comprising: OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, or IM-001. This composition may be administered together with a separate composition comprising the hPIV3 F protein antigen. The hPIV3 F protein antigen may comprise the amino acid sequence of SEQ ID NO: 3.

[0376] In some embodiments, the composition comprises a first mRNA and a second mRNA, wherein: (i) the first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2, wherein the first mRNA and the second mRNA are formulated into the same LNP comprising: 40% of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, or IM-001; 1.5% of DMG-PEG2000; 28.5% of cholesterol; and 30% of DOPE. This composition may be administered together with a separate composition comprising the hPIV3F protein antigen. The hPIV3 F protein antigen may contain the amino acid sequence of SEQ ID NO: 3.

[0377] In some embodiments, the composition comprises a first mRNA and a second mRNA, wherein: (i) the first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2, wherein the first mRNA and the second mRNA are formulated into separate LNPs comprising: 40% of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, or IM-001 in a molar ratio; 1.5% of DMG-PEG2000 in a molar ratio; 28.5% of cholesterol in a molar ratio; and 30% of DOPE in a molar ratio. This composition may be administered together with a separate composition comprising the hPIV3F protein antigen. The hPIV3 F protein antigen may contain the amino acid sequence of SEQ ID NO: 3.

[0378] In some embodiments, the composition comprises a first mRNA and a second mRNA, wherein: (i) the first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2, wherein the first mRNA and the second mRNA are formulated into the same LNP comprising GL-HEPES-E3-E12-DS-4-E10. This composition may be administered together with a separate composition comprising the hPIV3 F protein antigen, which may comprise the amino acid sequence of SEQ ID NO: 3.

[0379] In some embodiments, the composition comprises a first mRNA and a second mRNA, wherein: (i) the first mRNA encodes an hRSV F protein antigen comprising the amino acid sequence of SEQ ID NO: 1; and (ii) the second mRNA encodes an hMPV F protein antigen comprising the amino acid sequence of SEQ ID NO: 2, wherein the first mRNA and the second mRNA are formulated into separate LNPs comprising GL-HEPES-E3-E12-DS-4-E10. This composition may be administered together with a separate composition comprising the hPIV3 F protein antigen. The hPIV3 F protein antigen may comprise the amino acid sequence of SEQ ID NO: 3.

[0380] The composition in any of the above embodiments may comprise an hRSV F protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 1.

[0381] The composition in any of the above embodiments may comprise an hMPV F protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 2.

[0382] The composition in any of the above embodiments may comprise an hPIV3 F protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 3.

[0383] The composition in any of the above embodiments may comprise mRNA encoding the hRSV F protein antigen having a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4.

[0384] The composition in any of the above embodiments may comprise mRNA encoding the hMPV F protein antigen having a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 5.

[0385] The composition in any of the above embodiments may comprise mRNA encoding the hPIV3 F protein antigen having a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 6.

[0386] The composition in any of the above embodiments may contain at least one F protein antigenic polypeptide as a pre-fusion protein.

[0387] The composition in any of the above embodiments may contain at least one codon-optimized mRNA ORF.

[0388] The composition in any of the above embodiments may contain at least one mRNA, which contains at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR) and at least one polyadenylated (poly(A)) sequence.

[0389] The composition in any of the above embodiments may contain at least one mRNA, which contains a 5' UTR having a nucleic acid sequence having at least 80% identity with SEQ ID NO: 7.

[0390] The composition in any of the above embodiments may contain at least one mRNA, which contains a 3' UTR having a nucleic acid sequence having at least 80% identity with SEQ ID NO: 8.

[0391] The composition in any of the above embodiments may contain at least one mRNA having at least one chemical modification.

[0392] The composition in any of the above embodiments may comprise 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 chemically modified uracil nucleotides in at least one of these mRNAs.

[0393] The composition in any of the above embodiments may contain 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 chemically modified uracil nucleotides in at least one of these ORFs.

[0394] The composition in any of the above embodiments may include chemical modifications selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-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-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0395] The composition in any of the above embodiments may include chemical modifications selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0396] The composition in any of the above embodiments may contain chemically modified N1-methylpseuuridine.

[0397] The composition in any of the above embodiments may comprise mRNA encoding hRSV F protein antigen, mRNA encoding hMPV F protein antigen, and mRNA encoding hPIV3 F protein antigen that are not covalently linked to each other.

[0398] The composition in any of the above embodiments may comprise mRNAs encoding hRSV F protein antigen, hMPV F protein antigen, and / or hPIV3 F protein antigen, which are covalently linked to each other.

[0399] The compositions in any of the above embodiments may contain mRNA encoding hRSV F protein antigen, mRNA encoding hMPV F protein antigen, and mRNA encoding hPIV3 F protein antigen, each formulated into separate lipid nanoparticles (LNPs).

[0400] The composition in any of the above embodiments may contain mRNA encoding hRSV F protein antigen, mRNA encoding hMPV F protein antigen, and mRNA encoding hPIV3 F protein antigen formulated into the same LNP.

[0401] The composition in any of the above embodiments may contain LNP, wherein the LNP contains at least one cationic lipid.

[0402] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is biodegradable.

[0403] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is not biodegradable.

[0404] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is cleavable.

[0405] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is not cleavable.

[0406] The composition in any of the above embodiments may comprise LNP, wherein 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, GL-HEPES-E3-E12-DS-3-E14 and IM-001.

[0407] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is cKK-E10.

[0408] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

[0409] The composition in any of the above embodiments may contain LNP, wherein the cationic lipid is IM-001.

[0410] The composition in any of the above embodiments may contain LNP, wherein the LNP further comprises polyethylene glycol (PEG) conjugated (PEGylated) lipids, cholesterol-based lipids, and auxiliary lipids.

[0411] The composition in any of the above embodiments may comprise an LNP, wherein the LNP is composed of: 35% to 55% cationic lipids in a molar ratio; 0.25% to 2.75% polyethylene glycol (PEG) conjugated (PEGylated) lipids in a molar ratio; 20% to 45% cholesterol-based lipids in a molar ratio; and 5% to 35% auxiliary lipids in a molar ratio, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0412] The composition in any of the above embodiments may comprise an LNP, wherein the LNP is composed of: 40% cationic lipids; 1.5% PEGylated lipids; 28.5% cholesterol-based lipids; and 30% auxiliary lipids, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0413] The composition in any of the above embodiments may contain LNP, wherein the PEGylated lipid is dimyristic-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0414] The composition in any of the above embodiments may contain LNP, wherein the cholesterol-based lipid is cholesterol.

[0415] The composition in any of the above embodiments may contain LNP, wherein the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0416] The composition in any of the above embodiments may comprise an LNP, wherein the LNP is composed of: 40% GL-HEPES-E3-E12-DS-4-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0417] The composition in any of the above embodiments may comprise an LNP, wherein the LNP is composed of: 40% cKK-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0418] The composition in any of the above embodiments may comprise an LNP, wherein the LNP is composed of: 40% IM-001 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio, wherein all of these molar ratios are relative to the total lipid content of the LNP.

[0419] The composition in any of the above embodiments may contain LNPs, wherein the average diameter of the LNPs is from 30 nm to 200 nm.

[0420] The composition in any of the above embodiments may contain LNPs, wherein the average diameter of the LNPs is 80 nm to 150 nm.

[0421] Section VII of this specification further describes the LNPs that can be formulated together with the combined respiratory mRNA compositions discussed above and incorporated herein. VI.A. hRSV, hMPV and hPIV3 mRNA

[0422] As discussed in the examples, the compatibility of pre-fusion hRSV mRNA, pre-fusion hMPV mRNA, and pre-fusion hPIV3 mRNA was studied in vitro and in vivo. The studies demonstrated that when pre-fusion hRSV mRNA, pre-fusion hMPV mRNA, and pre-fusion hPIV3 mRNA were co-formulated or co-administered in a 1:1:1 ratio, there was inhibition of hMPV protein expression in vitro and hMPV immunogenicity in mice, relative to monovalent hMPV immunization. It was determined that interference between the components in the composition may occur at the level of mRNA expression in the absence of the immunodominant component.

[0423] To determine whether the inhibition of hMPV immunogenicity could be overcome by dose adjustment through altering the ratios of pre-fusion hRSV, hMPV, and hPIV3 mRNAs in co-prepared and co-administered formulations, various hRSV, hMPV, and hPIV3 ratios were investigated. Both co-prepared and co-administered studies demonstrated that increasing the hMPV mRNA dose overcame the interference without negatively impacting hRSV or hPIV3 immunogenicity, while decreasing the hPIV3 mRNA dose did not overcome the interference.

[0424] Therefore, the ratio of hRSV mRNA, hMPV mRNA and / or hPIV3 mRNA in combination vaccines is important for vaccine efficacy.

[0425] The composition in any of the above embodiments may comprise about 1:1:1, 1:1.1:1, 1:1.2:1, 1:1.3:1, 1:1.4:1, 1:1.5:1, 1:1.6:1, 1:1.7:1, 1:1.8:1, 1:1.9:1, 1:2:1, 1:2.1:1, 1:2.2:1, 1:2.3:1, 1:2.4:1, 1:2.5:1, 1:2.6:1, 1:2.7:1, 1:2.8:1, 1:2.9:1, 1: 3 : 1、1 : 3.1 : 1、1 : 3.2 : 1、1 : 3.3 : 1、1 : 3.4 : 1、1 : 3.5 : 1、1 : 3.6 : 1、1 : 3.7 : 1、1 : 3.8 :1、1 : 3.9 : 1、1 : 4 : 1、1 : 4.1 : 1、1 : 4.2 : 1、1 : 4.3 : 1、1 : 4.4 : 1、1 : 4.5 : 1、1 : 4.6 : 1、1 : 4.7 : 1、1 : 4.8 : 1、1 : 4.9 : 1、1 : 5 : 1、1 : 5.1 :1、1 : 5.2 : 1、1 : 5.3 : 1、1 : 5.4 : 1、1 : 5.5 : 1、1 : 5.6 : 1、1 : 5.7 : 1、1 : 5.8 : 1、1 : 5.9 : 1、1 : 6 : 1、1 : 6.1 : 1、1 : 6.2 : 1、1 : 6.3 : 1、1 : 6.4 :1、1 : 6.5 : 1、1 : 6.6 : 1、1 : 6.7 : 1、1 : 6.8 : 1、1 : 6.9 : 1、1 : 7 : 1、1 : 7.1 : 1、1 : 7.2 : 1、1 : 7.3 : 1、1 : 7.4 : 1、1 : 7.5 : 1、1 : 7.6 : 1、1 : 7.7 :1、1 : 7.8 : 1、1 : 7.9 : 1、1 : 8 : 1、1 : 8.1 : 1、1 : 8.2 : 1、1 : 8.3 : 1、1 : 8.4 : 1、1 : 8.5 : 1、1 : 8.6 : 1、1 : 8.7 : 1、1 : 8.8 : 1、1 : 8.9 : 1、1 : 9 :1、1 : 9.1 : 1、1 : 9.2 : 1、1 : 9.3 : 1, 1 : 9.The hRSV:hMPV:hPIV3 mRNA ratios (w / w) were 4:1, 1:9.5:1, 1:9.6:1, 1:9.7:1, 1:9.8:1, 1:9.9:1, 1:10:1, 1:1:0.9, 1:1:0.8, 1:1:0.7, 1:1:0.6, 1:1:0.5, 1:1:0.4, 1:1:0.3, 1:1:0.2, and 1:1:0.1.

[0426] The composition in any of the above embodiments may comprise 1:1:1, 1:1.1:1, 1:1.2:1, 1:1.3:1, 1:1.4:1, 1:1.5:1, 1:1.6:1, 1:1.7:1, 1:1.8:1, 1:1.9:1, 1:2:1, 1:2.1:1, 1:2.2:1, 1:2.3:1, 1:2.4:1, 1:2.5:1, 1:2.6:1, 1:2.7:1, 1:2.8:1, 1:2.9:1, 1:1:1. 3 : 1、1 : 3.1 : 1、1 : 3.2 : 1、1 : 3.3 : 1、1 : 3.4 : 1、1 : 3.5 : 1、1 : 3.6 : 1、1 : 3.7 : 1、1 : 3.8 :1、1 : 3.9 : 1、1 : 4 : 1、1 : 4.1 : 1、1 : 4.2 : 1、1 : 4.3 : 1、1 : 4.4 : 1、1 : 4.5 : 1、1 : 4.6 : 1、1 : 4.7 : 1、1 : 4.8 : 1、1 : 4.9 : 1、1 : 5 : 1、1 : 5.1 :1、1 : 5.2 : 1、1 : 5.3 : 1、1 : 5.4 : 1、1 : 5.5 : 1、1 : 5.6 : 1、1 : 5.7 : 1、1 : 5.8 : 1、1 : 5.9 : 1、1 : 6 : 1、1 : 6.1 : 1、1 : 6.2 : 1、1 : 6.3 : 1、1 : 6.4 :1、1 : 6.5 : 1、1 : 6.6 : 1、1 : 6.7 : 1、1 : 6.8 : 1、1 : 6.9 : 1、1 : 7 : 1、1 : 7.1 : 1、1 : 7.2 : 1、1 : 7.3 : 1、1 : 7.4 : 1、1 : 7.5 : 1、1 : 7.6 : 1、1 : 7.7 :1、1 : 7.8 : 1、1 : 7.9 : 1、1 : 8 : 1、1 : 8.1 : 1、1 : 8.2 : 1、1 : 8.3 : 1、1 : 8.4 : 1、1 : 8.5 : 1、1 : 8.6 : 1、1 : 8.7 : 1、1 : 8.8 : 1、1 : 8.9 : 1、1 : 9 :1、1 : 9.1 : 1、1 : 9.2 : 1、1 : 9.3 : 1, 1 : 9.The hRSV:hMPV:hPIV3 mRNA ratios (w / w) were 4:1, 1:9.5:1, 1:9.6:1, 1:9.7:1, 1:9.8:1, 1:9.9:1, 1:10:1, 1:1:0.9, 1:1:0.8, 1:1:0.7, 1:1:0.6, 1:1:0.5, 1:1:0.4, 1:1:0.3, 1:1:0.2, and 1:1:0.1.

[0427] The composition in any of the above embodiments may comprise about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1: 4, 1: 4.1, 1: 4.2, 1: 4.3, 1: 4.4, 1: 4.5, 1: 4.6, 1: 4.7, 1: 4.8, 1: 4.9, 1: 5, 1: 5.1, 1: 5.2, 1: 5.3, 1: 5.4, 1: 5.5, 1: 5.6, 1: 5.7, 1: 5.8, 1: 5.9, 1: 6, 1: 6.1, 1: 6.2, 1: 6.3, 1: 6.4, 1: 6.5, 1: 6.6, 1: 6.7, 1: 6.8, 1: 6.9, 1: 7, 1: 7.1, 1: The hRSV:hMPV mRNA ratios (w / w) of 7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, and 1:10.

[0428] The composition in any of the above embodiments may contain 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1: 4, 1: 4.1, 1: 4.2, 1: 4.3, 1: 4.4, 1: 4.5, 1: 4.6, 1: 4.7, 1: 4.8, 1: 4.9, 1: 5, 1: 5.1, 1: 5.2, 1: 5.3, 1: 5.4, 1: 5.5, 1: 5.6, 1: 5.7, 1: 5.8, 1: 5.9, 1: 6, 1: 6.1, 1: 6.2, 1: 6.3, 1: 6.4, 1: 6.5, 1: 6.6, 1: 6.7, 1: 6.8, 1: 6.9, 1: 7, 1: 7.1, 1: The hRSV:hMPV mRNA ratios (w / w) of 7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, and 1:10.

[0429] The composition in any of the above embodiments may comprise about 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1. 1.4.1 : 1.4.2 : 1.4.3 : 1.4.4 : 1.4.5 : 1.4.6 : 1.4.7 : 1.4.8 : 1.4.9 : 1.5 : 1.5.1 : 1.5.2 : 1.5.3 : 1.5.4 : 1.5.5 : 1.5.6 : 1.5.7 : 1.5.8 : 1.5.9 : 1.6 : 1.6.1 : 1.6.2 : 1.6.3 : 1.6.4 : 1.6.5 : 1.6.6 : 1.6.7 : 1.6.8 : 1.6.9 : 1.7 : 1.7.1 : 1.7.2 : 1、7.3 :1、7.4 : 1、7.5 : 1、7.6 : 1、7.7 : 1、7.8 : 1、7.9 : 1、8 : 1、8.1 : 1、8.2 : 1、8.3 :1、8.4 : 1、8.5 : 1、8.6 : 1、8.7 : 1、8.8 : 1、8.9 : 1、9 : 1、9.1 : 1、9.2 : 1、9.3 :1、9.4 : 1、9.5 : 1、9.6 : 1、9.7 : 1、9.8 : 1、9.9 : 1、10 : 1、1 : 0.9、1 : 0.8、1 :0.7、1 hMPV:hPIV3 mRNA ratios (w / w) of 0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, and 1:0.1.

[0430] The composition in any of the above embodiments may comprise 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4: 1.4.1 : 1.4.2 : 1.4.3 : 1.4.4 : 1.4.5 : 1.4.6 : 1.4.7 : 1.4.8 : 1.4.9 : 1.5 : 1.5.1 : 1.5.2 : 1.5.3 : 1.5.4 : 1.5.5 : 1.5.6 : 1.5.7 : 1.5.8 : 1.5.9 : 1.6 : 1.6.1 : 1.6.2 : 1.6.3 : 1.6.4 : 1.6.5 : 1.6.6 : 1.6.7 : 1.6.8 : 1.6.9 : 1.7 : 1.7.1 : 1.7.2 : 1、7.3 : 1、7.4 : 1、7.5 : 1、7.6 : 1、7.7 : 1、7.8 : 1、7.9 : 1、8 : 1、8.1 : 1、8.2 : 1、8.3 : 1、8.4 : 1、8.5 : 1、8.6 : 1、8.7 : 1、8.8 : 1、8.9 : 1、9 : 1、9.1 : 1、9.2 : 1、9.3 : 1、9.4 : 1、9.5 : 1、9.6 : 1、9.7 : 1、9.8 : 1、9.9 : 1、10 : 1、1 : 0.9、1 : 0.8、1 : 0.7、1 hMPV:hPIV3 mRNA ratios (w / w) of 0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, and 1:0.1.

[0431] The composition in any of the above embodiments may contain an hRSV:hMPV:hPIV3 mRNA ratio (w / w) of about 1:1:1 to about 1:10:1.

[0432] The composition in any of the above embodiments may contain an hRSV:hMPV:hPIV3 mRNA ratio expressed in micrograms (µg).

[0433] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 1 microgram of hMPV mRNA to about 1 microgram of hPIV3 mRNA.

[0434] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 2 micrograms of hMPV mRNA to about 1 microgram of hPIV3 mRNA.

[0435] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 3 micrograms of hMPV mRNA to about 1 microgram of hPIV3 mRNA.

[0436] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 5 micrograms of hMPV mRNA to about 1 microgram of hPIV3 mRNA.

[0437] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 0.5 μg hRSV mRNA to about 0.5 μg hMPV mRNA to about 0.1 μg hPIV3 mRNA.

[0438] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 0.5 μg hRSV mRNA to about 0.5 μg hMPV mRNA to about 0.5 μg hPIV3 mRNA.

[0439] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 0.5 μg hRSV mRNA to about 1.5 μg hMPV mRNA to about 0.5 μg hPIV3 mRNA.

[0440] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 0.5 micrograms of hRSV mRNA to about 1 microgram of hMPV mRNA to about 0.5 micrograms of hPIV3 mRNA.

[0441] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of about 0.5 μg hRSV mRNA to about 2.5 μg hMPV mRNA to about 0.5 μg hPIV3 mRNA.

[0442] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 1 microgram of hMPV mRNA to 1 microgram of hPIV3 mRNA.

[0443] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 2 micrograms of hMPV mRNA to 1 microgram of hPIV3 mRNA.

[0444] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 3 micrograms of hMPV mRNA to 1 microgram of hPIV3 mRNA.

[0445] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 1 μg hRSV mRNA to 5 μg hMPV mRNA to 1 μg hPIV3 mRNA.

[0446] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 0.5 μg hMPV mRNA to 0.1 μg hPIV3 mRNA.

[0447] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 0.5 μg hMPV mRNA to 0.5 μg hPIV3 mRNA.

[0448] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 1.5 μg hMPV mRNA to 0.5 μg hPIV3 mRNA.

[0449] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 1 μg hMPV mRNA to 0.5 μg hPIV3 mRNA.

[0450] The compositions in any of the above embodiments are wherein hRSV mRNA, hMPV mRNA and hPIV3 mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 2.5 μg hMPV mRNA to 0.5 μg hPIV3 mRNA.

[0451] The composition in any of the above embodiments may contain an hRSV:hMPV mRNA ratio (w / w) of about 1:1 to about 1:10.

[0452] The composition in any of the above embodiments may contain an hRSV:hMPV mRNA ratio expressed in micrograms (µg).

[0453] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 1 microgram of hMPV mRNA.

[0454] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 2 micrograms of hMPV mRNA.

[0455] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 3 micrograms of hMPV mRNA.

[0456] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 1 microgram of hRSV mRNA to about 5 micrograms of hMPV mRNA.

[0457] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 0.5 micrograms of hRSV mRNA to about 0.5 micrograms of hMPV mRNA.

[0458] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 0.5 micrograms of hRSV mRNA to about 1.5 micrograms of hMPV mRNA.

[0459] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 0.5 micrograms of hRSV mRNA to about 1 microgram of hMPV mRNA.

[0460] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of about 0.5 micrograms of hRSV mRNA to about 2.5 micrograms of hMPV mRNA.

[0461] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 1 microgram of hMPV mRNA.

[0462] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 2 micrograms of hMPV mRNA.

[0463] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 3 micrograms of hMPV mRNA.

[0464] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 1 microgram of hRSV mRNA to 5 micrograms of hMPV mRNA.

[0465] The compositions in any of the above embodiments, wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 0.5 μg hMPV mRNA.

[0466] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 1.5 μg hMPV mRNA.

[0467] The compositions in any of the above embodiments are wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 1 μg hMPV mRNA.

[0468] The compositions in any of the above embodiments, wherein hRSV mRNA and hMPV mRNA are present at a ratio (w / w) of 0.5 μg hRSV mRNA to 2.5 μg hMPV mRNA.

[0469] The composition in any of the above embodiments may comprise about 1:1:1, 1:1.1:1, 1:1.2:1, 1:1.3:1, 1:1.4:1, 1:1.5:1, 1:1.6:1, 1:1.7:1, 1:1.8:1, 1:1.9:1, 1:2:1, 1:2.1:1, 1:2.2:1, 1:2.3:1, 1:2.4:1, 1:2.5:1, 1:2.6:1, 1:2.7:1, 1:2.8:1, 1:2.9:1, 1: 3 : 1、1 : 3.1 : 1、1 : 3.2 : 1、1 : 3.3 : 1、1 : 3.4 : 1、1 : 3.5 : 1、1 : 3.6 : 1、1 : 3.7 : 1、1 : 3.8 :1、1 : 3.9 : 1、1 : 4 : 1、1 : 4.1 : 1、1 : 4.2 : 1、1 : 4.3 : 1、1 : 4.4 : 1、1 : 4.5 : 1、1 : 4.6 : 1、1 : 4.7 : 1、1 : 4.8 : 1、1 : 4.9 : 1、1 : 5 : 1、1 : 5.1 :1、1 : 5.2 : 1、1 : 5.3 : 1、1 : 5.4 : 1、1 : 5.5 : 1、1 : 5.6 : 1、1 : 5.7 : 1、1 : 5.8 : 1、1 : 5.9 : 1、1 : 6 : 1、1 : 6.1 : 1、1 : 6.2 : 1、1 : 6.3 : 1、1 : 6.4 :1、1 : 6.5 : 1、1 : 6.6 : 1、1 : 6.7 : 1、1 : 6.8 : 1、1 : 6.9 : 1、1 : 7 : 1、1 : 7.1 : 1、1 : 7.2 : 1、1 : 7.3 : 1、1 : 7.4 : 1、1 : 7.5 : 1、1 : 7.6 : 1、1 : 7.7 :1、1 : 7.8 : 1、1 : 7.9 : 1、1 : 8 : 1、1 : 8.1 : 1、1 : 8.2 : 1、1 : 8.3 : 1、1 : 8.4 : 1、1 : 8.5 : 1、1 : 8.6 : 1、1 : 8.7 : 1、1 : 8.8 : 1、1 : 8.9 : 1、1 : 9 :1、1 : 9.1 : 1、1 : 9.2 : 1、1 : 9.3 : 1, 1 : 9.The hRSV:hMPV:hPIV3 mRNA ratios (e.g., µg) were 4:1, 1:9.5:1, 1:9.6:1, 1:9.7:1, 1:9.8:1, 1:9.9:1, 1:10:1, 1:1:0.9, 1:1:0.8, 1:1:0.7, 1:1:0.6, 1:1:0.5, 1:1:0.4, 1:1:0.3, 1:1:0.2, and 1:1:0.1.

[0470] The composition in any of the above embodiments may comprise 1:1:1, 1:1.1:1, 1:1.2:1, 1:1.3:1, 1:1.4:1, 1:1.5:1, 1:1.6:1, 1:1.7:1, 1:1.8:1, 1:1.9:1, 1:2:1, 1:2.1:1, 1:2.2:1, 1:2.3:1, 1:2.4:1, 1:2.5:1, 1:2.6:1, 1:2.7:1, 1:2.8:1, 1:2.9:1, 1:1:1. 3 : 1、1 : 3.1 : 1、1 : 3.2 : 1、1 : 3.3 : 1、1 : 3.4 : 1、1 : 3.5 : 1、1 : 3.6 : 1、1 : 3.7 : 1、1 : 3.8 :1、1 : 3.9 : 1、1 : 4 : 1、1 : 4.1 : 1、1 : 4.2 : 1、1 : 4.3 : 1、1 : 4.4 : 1、1 : 4.5 : 1、1 : 4.6 : 1、1 : 4.7 : 1、1 : 4.8 : 1、1 : 4.9 : 1、1 : 5 : 1、1 : 5.1 :1、1 : 5.2 : 1、1 : 5.3 : 1、1 : 5.4 : 1、1 : 5.5 : 1、1 : 5.6 : 1、1 : 5.7 : 1、1 : 5.8 : 1、1 : 5.9 : 1、1 : 6 : 1、1 : 6.1 : 1、1 : 6.2 : 1、1 : 6.3 : 1、1 : 6.4 :1、1 : 6.5 : 1、1 : 6.6 : 1、1 : 6.7 : 1、1 : 6.8 : 1、1 : 6.9 : 1、1 : 7 : 1、1 : 7.1 : 1、1 : 7.2 : 1、1 : 7.3 : 1、1 : 7.4 : 1、1 : 7.5 : 1、1 : 7.6 : 1、1 : 7.7 :1、1 : 7.8 : 1、1 : 7.9 : 1、1 : 8 : 1、1 : 8.1 : 1、1 : 8.2 : 1、1 : 8.3 : 1、1 : 8.4 : 1、1 : 8.5 : 1、1 : 8.6 : 1、1 : 8.7 : 1、1 : 8.8 : 1、1 : 8.9 : 1、1 : 9 :1、1 : 9.1 : 1、1 : 9.2 : 1、1 : 9.3 : 1, 1 : 9.The hRSV:hMPV:hPIV3 mRNA ratios (e.g., µg) were 4:1, 1:9.5:1, 1:9.6:1, 1:9.7:1, 1:9.8:1, 1:9.9:1, 1:10:1, 1:1:0.9, 1:1:0.8, 1:1:0.7, 1:1:0.6, 1:1:0.5, 1:1:0.4, 1:1:0.3, 1:1:0.2, and 1:1:0.1.

[0471] The composition in any of the above embodiments may comprise about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1: 4, 1: 4.1, 1: 4.2, 1: 4.3, 1: 4.4, 1: 4.5, 1: 4.6, 1: 4.7, 1: 4.8, 1: 4.9, 1: 5, 1: 5.1, 1: 5.2, 1: 5.3, 1: 5.4, 1: 5.5, 1: 5.6, 1: 5.7, 1: 5.8, 1: 5.9, 1: 6, 1: 6.1, 1: 6.2, 1: 6.3, 1: 6.4, 1: 6.5, 1: 6.6, 1: 6.7, 1: 6.8, 1: 6.9, 1: 7, 1: 7.1, 1: 7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10 hRSV:hMPV mRNA ratios (e.g., µg).

[0472] The composition in any of the above embodiments may contain 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1: 4, 1: 4.1, 1: 4.2, 1: 4.3, 1: 4.4, 1: 4.5, 1: 4.6, 1: 4.7, 1: 4.8, 1: 4.9, 1: 5, 1: 5.1, 1: 5.2, 1: 5.3, 1: 5.4, 1: 5.5, 1: 5.6, 1: 5.7, 1: 5.8, 1: 5.9, 1: 6, 1: 6.1, 1: 6.2, 1: 6.3, 1: 6.4, 1: 6.5, 1: 6.6, 1: 6.7, 1: 6.8, 1: 6.9, 1: 7, 1: 7.1, 1: 7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10 hRSV:hMPV mRNA ratios (e.g., µg).

[0473] The composition in any of the above embodiments may comprise about 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1. 1.4.1 : 1.4.2 : 1.4.3 : 1.4.4 : 1.4.5 : 1.4.6 : 1.4.7 : 1.4.8 : 1.4.9 : 1.5 : 1.5.1 : 1.5.2 : 1.5.3 : 1.5.4 : 1.5.5 : 1.5.6 : 1.5.7 : 1.5.8 : 1.5.9 : 1.6 : 1.6.1 : 1.6.2 : 1.6.3 : 1.6.4 : 1.6.5 : 1.6.6 : 1.6.7 : 1.6.8 : 1.6.9 : 1.7 : 1.7.1 : 1.7.2 : 1、7.3 :1、7.4 : 1、7.5 : 1、7.6 : 1、7.7 : 1、7.8 : 1、7.9 : 1、8 : 1、8.1 : 1、8.2 : 1、8.3 :1、8.4 : 1、8.5 : 1、8.6 : 1、8.7 : 1、8.8 : 1、8.9 : 1、9 : 1、9.1 : 1、9.2 : 1、9.3 :1、9.4 : 1、9.5 : 1、9.6 : 1、9.7 : 1、9.8 : 1、9.9 : 1、10 : 1、1 : 0.9、1 : 0.8、1 :0.7、1 hMPV:hPIV3 mRNA ratios (e.g., µg) of 0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, and 1:0.1.

[0474] The composition in any of the above embodiments may comprise 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4: 1.4.1 : 1.4.2 : 1.4.3 : 1.4.4 : 1.4.5 : 1.4.6 : 1.4.7 : 1.4.8 : 1.4.9 : 1.5 : 1.5.1 : 1.5.2 : 1.5.3 : 1.5.4 : 1.5.5 : 1.5.6 : 1.5.7 : 1.5.8 : 1.5.9 : 1.6 : 1.6.1 : 1.6.2 : 1.6.3 : 1.6.4 : 1.6.5 : 1.6.6 : 1.6.7 : 1.6.8 : 1.6.9 : 1.7 : 1.7.1 : 1.7.2 : 1、7.3 : 1、7.4 : 1、7.5 : 1、7.6 : 1、7.7 : 1、7.8 : 1、7.9 : 1、8 : 1、8.1 : 1、8.2 : 1、8.3 : 1、8.4 : 1、8.5 : 1、8.6 : 1、8.7 : 1、8.8 : 1、8.9 : 1、9 : 1、9.1 : 1、9.2 : 1、9.3 : 1、9.4 : 1、9.5 : 1、9.6 : 1、9.7 : 1、9.8 : 1、9.9 : 1、10 : 1、1 : 0.9、1 : 0.8、1 : 0.7、1 hMPV:hPIV3 mRNA ratios of 0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, and 1:0.1 (e.g., µg).

[0475] The composition in any of the above embodiments may contain ratios expressed, for example, in picograms (pg), nanograms (ng), micrograms (μg), milligrams (mg).

[0476] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1 or about 1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0477] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.1 times or about 1.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0478] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.2 times or about 1.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0479] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.3 times or about 1.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0480] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.4 times or about 1.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0481] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.5 times or about 1.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0482] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.6 times or about 1.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0483] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.7 times or about 1.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0484] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.8 times or about 1.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0485] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 1.9 times or about 1.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0486] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be twice or about twice the amount of hRSV mRNA and / or hPIV3 mRNA.

[0487] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.1 times or about 2.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0488] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.2 times or about 2.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0489] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.3 times or about 2.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0490] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.4 times or about 2.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0491] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.5 times or about 2.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0492] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.6 times or about 2.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0493] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.7 times or about 2.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0494] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.8 times or about 2.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0495] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 2.9 times or about 2.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0496] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3 times or about 3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0497] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.1 times or about 3.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0498] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.2 times or about 3.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0499] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.3 times or about 3.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0500] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.4 times or about 3.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0501] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.5 times or about 3.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0502] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.6 times or about 3.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0503] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.7 times or about 3.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0504] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.8 times or about 3.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0505] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 3.9 times or about 3.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0506] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4 times or about 4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0507] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.1 times or about 4.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0508] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.2 times or about 4.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0509] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.3 times or about 4.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0510] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.4 times or about 4.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0511] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.5 times or about 4.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0512] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.6 times or about 4.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0513] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.7 times or about 4.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0514] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.8 times or about 4.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0515] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 4.9 times or about 4.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0516] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5 times or about 5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0517] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.1 times or about 5.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0518] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.2 times or about 5.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0519] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.3 times or about 5.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0520] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.4 times or about 5.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0521] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.5 times or about 5.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0522] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.6 times or about 5.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0523] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.7 times or about 5.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0524] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.8 times or about 5.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0525] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 5.9 times or about 5.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0526] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6 times or about 6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0527] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.1 times or about 6.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0528] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.2 times or about 6.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0529] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.3 times or about 6.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0530] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.4 times or about 6.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0531] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.5 times or about 6.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0532] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.6 times or about 6.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0533] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.7 times or about 6.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0534] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.8 times or about 6.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0535] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 6.9 times or about 6.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0536] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7 times or about 7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0537] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.1 times or about 7.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0538] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.2 times or about 7.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0539] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.3 times or about 7.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0540] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.4 times or about 7.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0541] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.5 times or about 7.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0542] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.6 times or about 7.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0543] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.7 times or about 7.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0544] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.8 times or about 7.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0545] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 7.9 times or about 7.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0546] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8 times or about 8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0547] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.1 times or about 8.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0548] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.2 times or about 8.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0549] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.3 times or about 8.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0550] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.4 times or about 8.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0551] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.5 times or about 8.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0552] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.6 times or about 8.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0553] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.7 times or about 8.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0554] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.8 times or about 8.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0555] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 8.9 times or about 8.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0556] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9 times or about 9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0557] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.1 times or about 9.1 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0558] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.2 times or about 9.2 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0559] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.3 times or about 9.3 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0560] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.4 times or about 9.4 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0561] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.5 times or about 9.5 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0562] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.6 times or about 9.6 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0563] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.7 times or about 9.7 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0564] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.8 times or about 9.8 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0565] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 9.9 times or about 9.9 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0566] The amount of hMPV mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 10 times or about 10 times the amount of hRSV mRNA and / or hPIV3 mRNA.

[0567] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.9 times or about 0.9 times the amount of hRSV mRNA and / or hMPV mRNA.

[0568] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.8 times or about 0.8 times the amount of hRSV mRNA and / or hMPV mRNA.

[0569] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.7 times or about 0.7 times the amount of hRSV mRNA and / or hMPV mRNA.

[0570] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.6 times or about 0.6 times the amount of hRSV mRNA and / or hMPV mRNA.

[0571] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.5 times or about 0.5 times the amount of hRSV mRNA and / or hMPV mRNA.

[0572] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.4 times or about 0.4 times the amount of hRSV mRNA and / or hMPV mRNA.

[0573] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.3 times or about 0.3 times the amount of hRSV mRNA and / or hMPV mRNA.

[0574] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.2 times or about 0.2 times the amount of hRSV mRNA and / or hMPV mRNA.

[0575] The amount of hPIV3 mRNA contained in the composition of any of the above embodiments (e.g., µg) may be 0.1 times or about 0.1 times the amount of hRSV mRNA and / or hMPV mRNA.

[0576] The composition in any of the above embodiments may contain an amount of mRNA, for example, expressed in picograms (pg), nanograms (ng), micrograms (μg), milligrams (mg). VII. Lipid nanoparticles (LNP)

[0577] The LNP disclosed herein may comprise four types of lipids: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids (e.g., cholesterol); and (iv) accessory lipids. VII.A. Cationic lipids

[0578] Ionizable lipids promote mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH to promote the efficient encapsulation of negatively charged mRNA drug substances. Exemplary cationic lipids are shown in Table 1 below.

[0579] Table 1 - Ionizable Lipids

[0580] Cationic lipids may be selected from the group consisting of: [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butyrate (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)butyryl)oxy)heptadecanoic acid ester (L319); 8-{(2-hydroxyethyl [6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid 9-heptadecyl ester (SM-102); [(4-hydroxybutyl)azanidinediyl]bis(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); N-[2-(dimethylamino)ethyl]carbamate [(3S,8S,9S,10R,13R,14S,17R)- 10,13-Dimethyl-17-[(2R)-6-methylheptane-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecano-1H-cyclopentadien[a]phenanthrene-3-yl] ester (DC-Chol); 3,3′,3′′,3′′′-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate tetra(8-methylnonyl) ester (306Oi10); (2-(dioctylammonium)ethyl)decyl phosphate (9A1P9); 5,5-di((Z)-heptadecane-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazolium -2-Carboxyethyl ester (A2-Iso5-2DC18); 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazatanediyl)diapropionate bis(2-(dodecyldithioyl)ethyl) ester (BAME-O16B); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)diaanediyl)bis(dodecane-2-ol) (C12-200); 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);9,9′,9′′,9′′′,9′′′′,9′′′′′-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))hexanonanoate hexa(octane-3-yl) ester (FTT5); (9Z,9′Z,9′′Z,9′′′Z,12Z,12′Z,12′′Z,12′′′Z)-tetra(octadecano-9,12-dienoic acid)(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(ethane-2,1-diyl) ester (OF-Deg-Lin); TT3; N; 1 N 3 N 5 - Tris(3-(bis(dodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxymethylamino)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoic acid heptadecanyl-9-yl ester (lipid 5); IM-001 and combinations thereof.

[0581] In some embodiments, the cationic lipid is biodegradable.

[0582] In various embodiments, the cationic lipid is not biodegradable.

[0583] In some embodiments, the cationic lipid is cleavable.

[0584] In some embodiments, the cationic lipid is not cleavable.

[0585] Cationic lipids are described in further detail in Dong et al. (PNAS. Proceedings of the National Academy of Sciences 111(11):3955-60. 2014); Fenton et al. (Adv. Mater. Advanced Materials 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. VII.B. PEGylated lipids

[0586] PEGylated lipid components offer control over the particle size and stability of nanoparticles. The addition of such components can prevent complex aggregation and provides a means to increase the cycle life of lipid-nucleic acid drug compositions and their delivery to target tissues (Klibanov et al., FEBS Letters [Federation of European Biochemical Societies Letters] 268(1):235-7. 1990). These components can be selected for rapid in vivo exchange of drug compositions (see, for example, U.S. Patent No. 5,885,613).

[0587] The PEGylated lipids under consideration include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length, which are covalently attached to C6-C6 bonds. 20 (For example, C8, C) 10 C 12 C 14 C 16 Or C 18 Lipids with one or more alkyl chains of length 1000, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipids are 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol (DMG-PEG); 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauryl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearyl-racemic-glycerol-polyethylene glycol (DSG-PEG); PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); and combinations thereof.

[0588] In some embodiments, the PEG has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipids described herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide). In some embodiments, the PEGylated lipids described herein are DMG-PEG2000. VII.C. Cholesterol-based lipids

[0589] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. 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-ethylformamidocholesterol), 1,4-bis(3-N-oleenylaminopropyl)piperazine (Gao et al., BiochemBiophys Res Comm. [Biochemistry and Biophysics Research Communications] (1991) 179:280; Wolf et al., BioTechniques [Biotechnology] (1997) 23:139; US Patent 5,744,335), and imidazole cholesterol ester (“ICE”; International Publication No. WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-stigmasterol, sitosterol, fucosterol, stigmasterol (stigmasterane-5,22-dien-3-ol), ergosterol; 3β-hydroxy-5,24-cholestadiene; lanosterol (8,24-lanosterdien-3β-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); yeast sterol (5α-cholest-8,24-dien-3β-ol); cholesterol Lathosterol (5α-cholesterol-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campesterol-5-en-3β-ol); campestanol (5α-campestanol-3β-ol); 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3β-ol); cholesterol margarate (cholesterol-5-en-3β-yl margarate); cholesterol oleate; cholesterol stearate; and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipids used in the LNP are cholesterol. VII.D. Helper lipids

[0590] The assisting lipid enhances the structural stability of the LNP and facilitates its escape from the endosome. It improves the uptake and release of the mRNA drug payload. In some embodiments, the assisting lipid is a zwitterionic lipid with fusion-promoting properties for enhancing the uptake and release of the drug payload. Examples of accessory lipids are 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE); 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC); 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS); 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine (DEPE); and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-distearylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE).

[0591] Other exemplary cofactor lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramides, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In some embodiments, the cofactor lipid is DOPE. In some embodiments, the cofactor lipid is DSPC.

[0592] In various embodiments, the LNP of the present invention 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. VII.E. Molar ratio of lipid components

[0593] The molar ratios of the above components are important for the effectiveness of LNP in delivering mRNA. The molar ratio of cationic lipids, PEGylated lipids, cholesterol-based lipids, and cofactor lipids is A : B : C : D, where A + B + C + D = 100%. In some embodiments, the molar ratio of cationic lipids to total lipids (i.e., A) in the LNP is 35%-55%, such as 35%-50% (e.g., 38%-42%, such as 40% or 45%-50%). In some embodiments, the molar ratio of the PEGylated lipid component to total lipids (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 lipids to total lipids (i.e., C) is 20%-50% (e.g., 27%-30%, such as 28.5% or 38%-43%). In some embodiments, the molar ratio of the auxiliary lipids to the total lipids (i.e., D) is 5%-35% (e.g., 28%-32%, such as 30% or 8%-12%, such as 10%). In some embodiments, the (PEGylated lipids + cholesterol) component has the same molar amount as the auxiliary lipids. In some embodiments, the molar ratio of the cationic lipids to the auxiliary lipids contained in the LNP is greater than 1.

[0594] In some embodiments, the LNP disclosed herein includes:

[0595] Cationic lipids with a molar ratio of 35% to 55% or 40% to 50% (e.g., cationic lipids with molar ratios of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% or 55%).

[0596] A polyethylene glycol (PEG) conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., PEGylated lipids in molar ratios of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%).

[0597] Cholesterol-based lipids with molar ratios of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., cholesterol-based lipids with molar ratios 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

[0598] The auxiliary lipids are in the molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., auxiliary lipids in the molar ratio of 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%).

[0599] All of these molar ratios are relative to the total lipid content of the LNP.

[0600] In some embodiments, the LNP comprises: 40% cationic lipids; 1.5% PEGylated lipids; 28.5% cholesterol-based lipids; and 30% auxiliary lipids.

[0601] In some embodiments, the PEGylated lipid is dimyristicoyl-PEG2000 (DMG-PEG2000).

[0602] In various embodiments, the cholesterol-based lipid is cholesterol.

[0603] In some embodiments, the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE).

[0604] In some embodiments, the LNP comprises: 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%.

[0605] In some embodiments, the LNP comprises: 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%.

[0606] In some embodiments, the LNP comprises: 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%.

[0607] In some embodiments, the LNP comprises: 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%.

[0608] In some embodiments, the LNP comprises: 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%.

[0609] In some embodiments, the LNP comprises: 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%.

[0610] In some embodiments, the LNP comprises: 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%.

[0611] In some embodiments, the LNP comprises: 40% OF-02 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio. This LNP formulation is designated herein as "Lipid A".

[0612] In some embodiments, the LNP comprises: 40% cKK-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio. Such LNP formulations are designated herein as "Lipid B".

[0613] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio. This LNP formulation is designated herein as "Lipid C".

[0614] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-4-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio. This LNP formulation is designated herein as "Lipid D".

[0615] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-3-E14 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio. This LNP formulation is designated herein as "Lipid E".

[0616] In some embodiments, the LNP comprises: 50% of 9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); 10% of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC); 38.5% of cholesterol; and 1.5% of 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000).

[0617] In some embodiments, the LNP comprises: 46.3% (4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 9.4% 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC); 42.7% cholesterol; and 1.6% 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0618] In some embodiments, the LNP comprises: 47.4% molar of [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 10% molar of 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC); 40.9% molar of cholesterol; and 1.7% molar of 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0619] In some embodiments, the LNP comprises: 35% to 55% IM-001; 0.25% to 2.75% DMG-PEG2000; 20% to 50% cholesterol; and 5% to 35% DOPE.

[0620] In some embodiments, the LNP comprises: 40% IM-001; 1.5% DMG-PEG2000; 28.5% cholesterol; and 30% DOPE.

[0621] To calculate the actual amount of each lipid to be incorporated into the LNP formulation, the molar amount of the cationic lipid is first 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 LNP. Next, the molar amount of each other lipid 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. VII.F. Buffer and other components

[0622] To stabilize nucleic acids and / or LNPs (e.g., to extend the shelf life of vaccine products), to facilitate the administration of LNP pharmaceutical compositions, and / or to enhance the in vivo expression of nucleic acids, nucleic acids and / or LNPs may be formulated in combination with one or more carriers, targeting ligands, stabilizing agents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients include parabens, thimerosal, sodium thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0623] The LNP compositions disclosed herein can be provided in either a cryo-liquid or lyophilized form. Various cryoprotectants can be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextrose, etc. The cryoprotectant can constitute 5%-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, 5%-30% (e.g., 10%) (w / v). Once formulated with a cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at -20°C to -80°C.

[0624] The LNP composition can be provided to the patient in an aqueous buffer solution: if previously frozen, it is thawed, or if previously lyophilized, it is reconstituted at the bedside in an aqueous buffer solution. The buffer solution can be isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate-buffered saline (PBS). VIII. Methods for manufacturing LNP vaccines

[0625] The LNPs of this invention can be prepared using a variety of techniques. For example, multilayer vesicles (MLVs) can be prepared using conventional techniques, such as by depositing selected lipids onto the inner wall of a suitable container or vessel (by dissolving the lipids in a suitable solvent and then evaporating the solvent to leave a thin film inside the vessel) or by spray drying. An aqueous phase can then be added to the vessel accompanied by vortexing, which allows the MLV to form. Unilayer vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilayer vesicles. Alternatively, ULVs can be formed using detergent removal techniques.

[0626] Several methods are described in patent application publications US 2011 / 0244026, US 2016 / 0038432, US 2018 / 0153822, US 2018 / 0125989, and US 2021 / 0046192, and can be used to prepare LNP vaccines. One exemplary method requires encapsulating mRNA by mixing it with a mixture of lipids without first pre-forming the lipids into lipid nanoparticles, as described in patent application publication US 2016 / 0038432. Another exemplary method requires encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in patent application publication US 2018 / 0153822.

[0627] In some embodiments, a method for preparing LNPs loaded with mRNA includes heating one or more solutions to a temperature above ambient temperature, wherein the one or more solutions are a solution comprising pre-formed lipid nanoparticles, a solution comprising mRNA, and a mixed solution comprising LNP-encapsulated mRNA. In some embodiments, the method includes heating one or both of the mRNA solution and the pre-formed LNP solution prior to a mixing step. In some embodiments, the method includes heating one or more of the solution comprising the pre-formed LNPs, the solution comprising mRNA, and the solution comprising LNP-encapsulated mRNA during a mixing step. In some embodiments, the method includes heating the LNP-encapsulated mRNA after a mixing step. In some embodiments, the temperature to which the one or more solutions are heated is or greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, one or more solutions are heated to a temperature range of about 25°C-70°C, about 30°C-70°C, about 35°C-70°C, about 40°C-70°C, about 45°C-70°C, about 50°C-70°C, or about 60°C-70°C. In some embodiments, the temperature is about 65°C.

[0628] Various methods can be used to prepare mRNA solutions suitable for this disclosure. In some embodiments, mRNA can be directly dissolved in the buffer solution described herein. In some embodiments, the mRNA solution can be generated by mixing the mRNA stock solution with the buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, the mRNA solution can be generated by mixing the mRNA stock solution with the buffer solution immediately prior to mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA at a concentration in water or buffer 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.

[0629] In some embodiments, a pump is used to mix the mRNA stock solution with the buffer solution. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate of at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x that 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.

[0630] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 ml / min (e.g., about 5-50 ml / min, about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 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.

[0631] The process of incorporating desired mRNA into lipid nanoparticles is referred to as “addition”. An exemplary method is described in Lasic et al., FEBS Lett. [FEBS Communications] (1992) 312:255-8. The nucleic acid incorporated into the LNP can be entirely or partially located within the internal space of the lipid nanoparticle, within the bilayer of the lipid nanoparticle membrane, or associated with the outer surface of the lipid nanoparticle membrane. Incorporating mRNA into lipid nanoparticles is also referred to herein as “encapsulation,” in which the nucleic acid is completely or substantially contained within the internal space of the lipid nanoparticle.

[0632] Suitable LNPs can be prepared in various sizes. In some embodiments, reduced lipid nanoparticle size is associated with more efficient mRNA delivery. The selection of an appropriate LNP size can take into account the target cell or tissue site and, to some extent, the application for which the lipid nanoparticles will be used.

[0633] Various methods can be used to modify the size of lipid nanoparticle populations. In various embodiments, the method described herein utilizes a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of an LNP sample is mixed with 990 μl of 10% trehalose. This solution is placed in a cuvette and then placed in the Zetasizer machine. The z-mean diameter (nm), or cumulative 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 cumulative analysis of autocorrelation functions. The average LNP diameter can be reduced by sonicating the formed LNPs. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) assessments to guide efficient lipid nanoparticle synthesis.

[0634] In some embodiments, the majority of purified LNPs (i.e., LNPs with a purity greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) have a size of about 70–150 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., more than 80% or 90%) of the purified lipid nanoparticles are about 70-150 nm in size (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).

[0635] In some embodiments, the average diameter of the LNP is 30-200 nm.

[0636] In various embodiments, the average diameter of the LNP is 80-150 nm.

[0637] In some embodiments, the average size of the LNP in the composition of the present invention is 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.

[0638] In some embodiments, the size of more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs in the compositions of the present invention ranges from 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), which is particularly suitable for lung delivery via nebulization.

[0639] In some embodiments, the dispersibility or molecular size heterogeneity measure (PDI) of LNPs in the pharmaceutical compositions provided by this disclosure is less than about 0.5. In some embodiments, the PDI of LNPs is 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 using a Zetasizer machine as described above.

[0640] 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 compositions encapsulate mRNA within each individual particle. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 50% to 99%; or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%. Typically, the encapsulation efficiency of the lipid nanoparticles used herein is at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%).

[0641] In some embodiments, the N / P ratio of the LNP is from 1 to 10. In some embodiments, the N / P ratio of the lipid nanoparticles is greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In some embodiments, the typical N / P ratio of the LNPs described herein is 4.

[0642] In some embodiments, the pharmaceutical composition according to this 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, the pharmaceutical composition contains about 0.1 μg to 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.

[0643] In some embodiments, mRNA can be prepared by chemical synthesis or by in vitro transcription (IVT) of a DNA template. An exemplary method for preparing and purifying mRNA is described in Example 1. In this method, during IVT, a cDNA template is used to generate mRNA transcripts, and the DNA template is degraded by a DNase. The transcripts are purified by deep filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding a cap and tail, and the modified RNA is purified again by deep filtration and TFF.

[0644] The mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNP. The amphiphilic 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 3.0–7.0 (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 may contain other components, such as salts (e.g., sodium, potassium, and / or calcium salts). In a particular embodiment, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0645] An exemplary non-limiting method for preparing mRNA-LNP compositions involves mixing a buffered mRNA solution with an ethanol solution of lipids in a controlled, homogeneous manner, wherein the lipid:mRNA ratio is maintained throughout the mixing process. In this illustrative example, the mRNA is presented in an aqueous buffer containing citrate monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipids, PEGylated lipids, cholesterol-based lipids, and accessory lipids) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed at a 4:1 volume ratio in a T-shaped mixer with a near-pulseless pump system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be performed using a dialysis cassette or a TFF system. A TFF can be used to concentrate and buffer exchange the resulting nascent LNPs immediately after formation via the T-mixing process. The percolation process is a continuous operation, and the volume is kept constant by adding an appropriate buffer at the same rate as the percolation stream. Packaging and Use of IX.mRNA-LNP Vaccine

[0646] 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, mRNA-LNP vaccines can be formulated or packaged for pulmonary administration. In various embodiments, mRNA-LNP vaccines can be formulated or packaged for intravenous administration. The vaccine composition can be in the form of a ready-to-use formulation, wherein the LNP composition is lyophilized and reconstituted with physiological buffer (e.g., PBS) just before use. The vaccine composition can also be shipped and provided as an aqueous solution or a frozen aqueous solution and can be administered directly to the subject without reconstitution (or after thawing if previously frozen).

[0647] Therefore, this disclosure provides an article of manufacture (such as a kit) that provides an mRNA-LNP vaccine in a single container, or provides an mRNA-LNP vaccine in one container (e.g., a first container) and physiological buffer for reconstitution in another container (e.g., a second container). The one or more containers may contain a single-use dose or a multiple-use dose. The one or more containers may be pretreated glass vials or ampoules. The article of manufacture may also include instructions for use.

[0648] In certain embodiments, the mRNA-LNP vaccine is provided for use in an intramuscular (IM) injection. The vaccine may be injected into the deltoid muscle of the subject, for example, in his / her upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or syringe (e.g., single-chambered or multi-chambered). In some embodiments, the vaccine is provided for use in inhalation and is provided in a pre-filled pump, nebulizer, or inhaler.

[0649] In some embodiments, the nucleic acids, peptides, and compositions of the present invention are provided for use in skin injections, such as in the epidermis, dermis, or subcutaneous tissue of the skin. In some embodiments, the nucleic acids, peptides, and compositions are provided in a device suitable for skin injection, such as a needle (e.g., an epidermal, dermal, or subcutaneous tissue needle), a needle-free device, a microneedle device, or a microprotrusion array device. Examples of microneedle or microprotrusion array devices suitable for skin injection according to the present invention are described in US20230270842A1, US20220339416A1, US20210085598A1, US20200246450A1, US20220143376A1, US20180264244A1, US20180263641A1, and US20110245776A1.

[0650] mRNA-LNP vaccines can be administered to subjects in need at a preventative effective dose, which is an amount that provides sufficient immune protection against the target pathogen for a sustained duration (e.g., one year, two years, five years, ten years, or a lifetime). Sufficient immune protection can be, for example, prevention or reduction of symptoms associated with pathogen infection. In some embodiments, multiple doses (e.g., two doses) of the vaccine are administered (e.g., by injection) to subjects in need to achieve the desired preventative effect. The doses (e.g., a primary dose and a booster dose) can be spaced at intervals of at least, for example, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, or ten years. X. Carrier

[0651] In one aspect, this document discloses vectors including the mRNA compositions disclosed herein. RNA sequences encoding target proteins (e.g., mRNA encoding RSV F, hMPV F, or hPIV3 F proteins) can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and granules. Vectors of particular interest may include expression vectors, replication vectors, probe-generating vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0652] In some 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 the optimally translated IVT mRNA for therapeutic use is disclosed in detail in Sahin et al. (2014). Nat. Rev. Drug Discov. 13, 759–780; Weissman (2015). Expert Rev. Vaccines 14, 265–281.

[0653] In some embodiments, the vectors disclosed herein may include at least the following from 5' to 3': 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 contain polynucleotide sequences encoding a poly(A) sequence and / or a polyadenylation signal.

[0654] Various RNA polymerase promoters are known. In some embodiments, the promoter may be the T7 RNA polymerase promoter. Other useful promoters may include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Common nucleotide sequences for the T7, T3, and SP6 promoters are known.

[0655] This article also discloses host cells (e.g., mammalian cells, such as human cells) including the vectors or RNA compositions disclosed herein.

[0656] A variety of different methods, such as any of the commercially available methods, can be used to introduce polynucleotides into target cells. These methods include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipid transfection, polymer encapsulation, peptide-mediated transfection, and bioprojectile particle delivery systems (such as “gene guns”) (see, for example, Nishikawa et al. (2001). Hum Gene). Ther. [Human Gene Therapy] 12(8): 861-70 or TransIT-RNA Transfection Kit (Mirus, Madison, WI).

[0657] 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 used as a delivery carrier in vitro and in vivo is a liposome (e.g., an artificial membrane capsule).

[0658] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors disclosed herein, various measurements can be performed to confirm the presence of mRNA sequences in host cells. XI. Self-replicating RNA and trans-replicating RNA

[0659] Self-replicating RNA:

[0660] On the one hand, this article discloses self-replicating RNAs encoding RSV F protein, hMPV F protein and / or hPIV3 protein.

[0661] Self-replicating RNA can be generated by using replication elements derived from, for example, alphaviruses and replacing structural viral proteins with nucleotide sequences encoding target proteins (e.g., RSV F protein, hMPV F protein, hPIV3 F protein). Self-replicating RNA is typically a positive-strand molecule that can be directly translated upon delivery to the cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, along with collinear subgenomic transcripts, can be self-translated to provide in situ expression of the encoded antigens (i.e., RSV F protein antigen, hMPV F protein antigen, hPIV3 F protein antigen), or they can be transcribed to provide further transcripts with the same significance as the delivered RNA, which are then translated to provide in situ expression of the antigens. The overall result of this transcriptional sequence is a significant amplification of the number of introduced replicant RNAs, and thus the encoded antigens become the major polypeptide products of the cell.

[0662] A suitable system for self-replication in this manner is the use of alphavirus-based replicons. These replicons are positive-strand (sense-positive) RNAs that, upon delivery to the cell, lead to the translation of a replicase (or replicase-transcriptionase). The replicase is translated into a polyprotein, which automatically cleaves to provide a replication complex that produces a genomic strand copy of the positive-strand delivered RNA. These negative-strand transcripts can themselves be transcribed to produce further copies of the positive-strand parent RNA and also subgenomic transcripts encoding the antigen. Thus, the translation of the subgenomic transcripts results in the infected cell expressing the antigen in situ. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki forest virus, eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: International Publication No. WO 2005 / 113782, which is incorporated herein by reference.

[0663] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule, and (ii) an RSV F protein antigen, hMPV F protein antigen, or hPIV3 F protein antigen. The polymerase may be an alphavirus replicase, for example, including one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Although the native alphavirus genome encodes structural virion proteins in addition to non-structural replicase polyproteins, in some embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Therefore, the self-replicating RNA may result in the production of copies of its own genomic RNA within the cell, but not in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphavirus, the self-replicating RNA molecule cannot permanently persist itself in an infectious form. The alphavirus structural proteins necessary for permanent persistence in wild-type virus are not present in the self-replicating RNA disclosed herein, and their positions are replaced by one or more genes encoding a target immunogen, such that the subgenomic transcript encodes an immunogen, rather than an alphavirus structural virion protein. The self-replicating RNA is described in further detail in International Publication No. WO 2011 / 005799, which is incorporated herein by reference.

[0664] Trans-replicating RNA:

[0665] On one hand, this article discloses trans-replicating RNAs encoding RSV F protein, hMPV F protein, and / or hPIV3 protein.

[0666] Trans-replicating RNA has elements similar to those of the self-replicating RNA described above. However, for trans-replicating RNA, two separate RNA molecules are used. The first RNA molecule encodes the aforementioned RNA replicase (e.g., alphavirus replicase), and the second RNA molecule encodes the target protein (e.g., RSV F protein antigen, hMPV F protein antigen, hPIV3 F protein antigen). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby significantly increasing the copy number of the RNA molecule encoding the target protein. Trans-replicating RNA is further described in detail in International Publication No. WO 2017 / 162265, which is incorporated herein by reference. XII. Pharmaceutical Composition

[0667] The purified RNA according to this disclosure can be used as a component in pharmaceutical compositions, for example, as vaccines. These compositions will typically contain RNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions disclosed herein may also contain one or more additional components, such as small molecule immune enhancers (e.g., TLR agonists). The pharmaceutical compositions disclosed herein may also contain a delivery system for RNA, such as liposomes, oil-in-water emulsions, or microparticles. In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNPs). In some embodiments, the composition comprises a nucleic acid molecule encoding an antigen encapsulated within an LNP. XIII. Vaccination Methods

[0668] The combined respiratory mRNA vaccine compositions disclosed herein can be administered to subjects to induce an immune response against hRSV F protein, hMPV F protein, and / or hPIV3 protein, wherein the anti-antigen antibody titer in the subjects after vaccination is increased relative to the anti-antigen antibody titer in subjects not vaccinated with the compositions disclosed herein or relative to alternative vaccines against hRSV, hMPV, and / or hPIV3. "Anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0669] In one aspect, this disclosure provides a method for inducing an immune response to hRSV or a method for protecting a subject from hRSV infection, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method for inducing an immune response to hMPV or a method for protecting a subject from hMPV infection, the method comprising administering the composition described herein to the subject. In yet another aspect, this disclosure provides a method for inducing an immune response to hPIV3 or a method for protecting a subject from hPIV3 infection, the method comprising administering the composition described herein to the subject.

[0670] This disclosure provides compositions for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering the compositions described herein to the subject. This disclosure also provides compositions described herein for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering the compositions described herein to the subject. This disclosure further provides compositions described herein for use in inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the use comprising administering the compositions described herein to the subject.

[0671] This disclosure provides the compositions described herein for use in the manufacture of medicaments for inducing an immune response to hRSV or protecting a subject from hRSV infection. This disclosure also provides the compositions described herein for use in the manufacture of medicaments for inducing an immune response to hMPV or protecting a subject from hMPV infection. This disclosure further provides the compositions described herein for use in the manufacture of medicaments for inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection.

[0672] In some embodiments, the compositions described herein increase the serum concentration of antibodies that bind specifically to the site Ø of the RSV F protein. In some embodiments, the compositions described herein increase the serum concentration of antibodies that bind specifically to the site Ø of the hMPV F protein. In some embodiments, the compositions described herein increase the serum concentration of antibodies that bind specifically to the site Ø of the hPIV3 F protein.

[0673] In some embodiments, after administration of the composition described herein, the subject has substantially the same or higher serum concentration of anti-RSV neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO: 1. In some embodiments, after administration of the composition described herein, the subject has substantially the same or higher serum concentration of anti-hMPV neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO: 2. In some embodiments, after administration of the composition described herein, the subject has substantially the same or higher serum concentration of anti-hPIV3 neutralizing antibodies compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hPIV3 F protein antigen of SEQ ID NO: 3.

[0674] In some embodiments, after administration of the composition described herein, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies compared to a subject administered a protein hRSV vaccine. In some embodiments, after administration of the composition described herein, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies compared to a subject administered a protein hMPV vaccine. In some embodiments, after administration of the composition described herein, the subject has a comparable serum concentration of anti-hPIV3 neutralizing antibodies compared to a subject administered a protein hPIV3 vaccine.

[0675] In some embodiments, the compositions described herein increase the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hRSV. In some embodiments, the compositions described herein increase the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV. In some embodiments, the compositions described herein increase the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hPIV3. Example

[0676] The foregoing description of specific embodiments so fully discloses the general nature of this disclosure that others, by applying knowledge within the scope of the art, can readily modify and / or adapt such particular embodiments for various applications without excessive experimentation and without departing from the overall concept of this disclosure. Therefore, based on the teachings and guidance provided herein, such modifications and adaptations are intended to be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive and not limiting purposes, and therefore the terminology or terminology of this specification will be interpreted by those skilled in the art based on the teachings and guidance. Example 1: mRNAs encoding hRSV F protein, hMPV F protein, and hPIV3 F protein

[0677] RSV F, hMPV F, and hPIV3 F proteins were selected as candidates for testing a combined mRNA-based vaccine. The F protein designated as RSV FD1 corresponds to the WT RSV F protein. The F protein designated as RSV FD3 corresponds to the pre-fusion RSV F protein. The F protein designated as hMPV FD1 corresponds to the WT hMPV F protein. The F protein designated as hMPV FD2 corresponds to the pre-fusion hMPV F protein. The F protein designated as hPIV3 FD1 corresponds to the WT hPIV3 F protein. The F protein designated as hPIV3 FD2 corresponds to the pre-fusion hPIV3 F protein. The amino acid sequence of each F protein is listed below.

[0678] hRSV FD1: (SEQ ID NO: 9)

[0679] hRSV FD3: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNPETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKNGSNICLTRTDRGWYCDNAGNVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNELINQSLAFINQSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN(SEQ ID NO: 1)

[0680] hMPV FD1: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN (SEQ ID NO:10)

[0681] hMPV FD2: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTvVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEV TAIKNALKTTNEAVSTLGNGVRVLAfAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIY MVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLN KGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN (SEQ ID NO: 2) (Lowercase amino acids represent linker, fold motif, linker, HRV-3C cleavage site, linker, 8X-His tag and strep tag II region.)

[0682] hPIV3 FD1: MLISILSIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQAKSDIEKLKEAIRDTNKAVQSVQSSVGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGSWHQSSTTIIIILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK (SEQ ID NO:11)

[0683] hPIV3 FD2: MLISILSIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVAL VEAKQAKSDIEKLKEAIRDTNKAVQSVCSSVGNCIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNCFGDNIGSLQEKGIKLQCIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDV DLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTTCTCNGIGNRINQP PDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKVKSDLEESKEWYRRSNQKLDSIGSWHQSSTTIIIILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK (SEQ ID NO:3)

[0684] The mRNA described herein comprises an open reading frame (ORF) encoding the F protein antigen, at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and at least one polyadenylated (poly(A)) sequence. The mRNA further comprises a 5' cap having the following structure:

[0685] The following lists the nucleic acid sequences in the mRNA open reading frames (ORFs) encoding the hRSV FD3, hMPV FD2, and hPIV3 FD2 proteins.

[0686] hRSV FD3 mRNA ORF:

[0687] hMPV FD2 mRNA ORF ： (SEQ ID NO:5)

[0688] hPIV3 FD2 mRNA ORF : (SEQ ID NO:6)

[0689] The following lists the nucleic acid sequences in the DNA templates encoding codon-optimized hRSV FD3, hMPV FD2, and hPIV3 FD2 mRNA.

[0690] hRSV FD3 DNA:

[0691] hMPV FD2 DNA: (SEQ ID NO:13)

[0692] hPIV3 FD2 DNA: (SEQ ID NO: 14)

[0693] The following lists the nucleic acid sequences of 5' UTR and 3' UTR (regarding hRSV F, hMPV F, and hPIV3 F).

[0694] 5' UTR: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO:7)

[0695] 3' UTR: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCCUGGCCCUGGAAGUUGCCACACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 8)

[0696] The following lists the nucleic acid sequences of each of the full-length mRNAs encoding the proteins hRSV FD3, hMPV FD2, and hPIV3 FD2.

[0697] hRSV FD3 mRNA: (SEQ ID NO:15)

[0698] hMPV FD2 mRNA: (SEQ ID NO: 16)

[0699] hPIV3 FD2 mRNA: (SEQ ID NO: 17) Example 2: Compatibility study of hRSV, hMPV and hPIV3 mRNAs before fusion

[0700] The compatibility of the pre-fusion hRSV F, hPIV3 F, and hMPV F proteins listed above, when co-formulated or co-administered in a 1:1:1 ratio, was tested in vitro in human skeletal muscle (hSKM) cells and in vivo in mice. The full-length forms of the pre-fusion hRSV, pre-fusion hPIV3, and pre-fusion hMPV proteins were synthesized as mRNA and formulated as monovalent mRNA-LNP formulations and trivalent mRNA-LNP co-formulations in a 1:1:1 ratio. To test the co-administration of the pre-fusion hRSV, hMPV, and hPIV3 proteins in vivo, the monovalent mRNA-LNP formulations of each immunogen were combined in a 1:1:1 ratio prior to immunization to produce a mixture. Unless otherwise specified, the mRNA constructs were formulated with LNP OF-02.

[0701] For in vitro studies, hSKM cells were transfected with a trivalent hRSV:hMPV:hPIV3 mRNA-LNP co-formulation and monovalent mRNA-LNP formulations of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA at doses ranging from 0.088 µg to 3 µg. Flow cytometry was used to determine the inter-component interference of each formulation by assessing the expression levels of pre-fusion F-specific epitopes and common region epitopes, and these expression levels were calculated as integrated mean fluorescence intensity (iMFI). Transfection with monovalent mRNA-LNP formulations of pre-fusion hRSV F, hMPV F, and hPIV3 F resulted in robust dose-specific expression with similar patterns for both pre-fusion F epitopes and common region epitopes, indicating that the pre-fusion F monoclonal antibody binding site was well preserved in all three constructs after mRNA expression. Transfection with the trivalent mRNA-LNP co-formulation produced an expression pattern similar to that after monovalent transfection with RSV and PIV3, such as... Figures 1A-1D As shown in the figure. However, at higher dose levels, the expression of the hMPVF epitope in cells transfected with the trivalent mRNA-LNP co-formulation was lower than that in cells transfected with the monovalent formulation, as shown in the figure. Figure 1E As shown in the diagram. For example, at a dose of 3 µg, the iMFI of the pre-fusion hMPV F epitope in the trivalent mRNA-LNP co-formulation was 3-fold lower than that of the monovalent hMPV mRNA-LNP formulation. The iMFI of the hMPV common region epitope in the trivalent mRNA-LNP co-formulation was 2-fold lower than that of the monovalent hMPV mRNA-LNP formulation, as shown in the diagram. Figure 1F As shown in the image.

[0702] For in vivo studies, interference was determined by comparing the immunogenicity of mice vaccinated with a trivalent mRNA-LNP co-formulation (each dose containing 0.5 µg of pre-fusion hRSV mRNA, 0.5 µg of pre-fusion hPIV3 mRNA, and 0.5 µg of pre-fusion hMPV mRNA) or a mixture of three monovalent mRNA-LNP formulations (containing 0.5 µg / dose of pre-fusion hRSV mRNA, 0.5 µg / dose of pre-fusion hPIV3 mRNA, and 0.5 µg / dose of pre-fusion hMPV mRNA) with that of mice vaccinated with only a monovalent mRNA-LNP formulation (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). The titer of binding antibodies was determined by ELISA using plates coated with pre-fusion hRSV, hPIV3, or hMPV. Neutralizing antibody titers were determined by microneutralization against hRSV, hPIV3, or hMPV expressing green fluorescent protein (GFP) reporter antibodies. Similar levels of binding antibody titers and neutralizing antibody titers against RSV and hPIV3 were induced using all three immunization regimens (i.e., co-formulation, mixture, and monovalent regimens). Figures 2A-2D As shown in the figure. However, compared with the hMPV binding antibody titer induced by monovalent hMPV immunization, the hMPV binding antibody titer induced by the trivalent mRNA-LNP co-formulation was 2.7-fold lower (p < 0.001), and the titer of the mixture of the three monovalent mRNA-LNP formulations was 2.0-fold lower (p = 0.0096), as shown in the figure. Figure 2E As shown in the figure. Similarly, compared with the neutralizing antibody titers induced by immunization with monovalent hMPV, the hMPV-binding antibody titers induced by the trivalent mRNA-LNP co-formulation were 4.9-fold lower (p < 0.001), and the titers induced by the mixture of the three monovalent mRNA-LNP formulations were 2.6-fold lower (p = 0.0107), as shown in the figure. Figure 2F As shown in the image.

[0703] Data from the above studies demonstrate that when pre-fusion hRSV mRNA, pre-fusion hMPV mRNA, and pre-fusion hPIV3 mRNA are co-formulated or co-administered in a 1:1:1 ratio, compared to monovalent hMPV immunization, there is inhibition of hMPV protein expression in vitro and hMPV immunogenicity in mice. No effect on hRSV and hPIV3 protein expression or immunogenicity was shown. Example 3: Trivalent mixture study and hMPV immunogenicity

[0704] To determine whether the inhibition of hMPV immunogenicity was primarily due to decreased protein expression observed in vitro or the presence of additional immunodominant components, the compatibility of three immunogens (i.e., hRSV, hMPV, and hPIV3) as recombinant proteins was evaluated. After immunization of mice with a 1:1:1 mixture of adjuvanted recombinant proteins, the titers of binding antibodies induced against hRSV and hMPV were not significantly different from those induced by immunization with monovalent protein controls, while the titer of binding antibodies against hPIV3 was 3.3-fold lower than that in the group receiving hPIV3 alone (p = 0.0118). Figures 3A-3C As shown in the figure. However, there was no significant difference in the induction of neutralizing antibodies against any target between the protein mixture immunization group and the monovalent protein immunization group, as shown in the figure. Figures 3D-3F As shown in the figure. Data from this study demonstrate that interference may occur at mRNA expression levels where no immunodominant component is present.

[0705] To determine whether the inhibition of hMPV immunogenicity could be overcome by dose adjustments made by changing the ratio of pre-fusion hRSV, hMPV, and hPIV3 mRNA in the mixture, two methods were tested: (1) increasing the hMPV dose by 2 or 5-fold relative to the hRSV and hPIV3 doses; and (2) decreasing the hPIV3 dose by 5-fold relative to the hRSV and hMPV doses, as shown in Table 1 below. Unless otherwise specified, the mRNA constructs were formulated with LNP OF-02. Table 1 - Dosage / Ratio of hRSV, hMPV, and hPIV3 mRNA before mixture fusion, as tested with LNP OF-02

[0706] The effect of increasing hMPV dose in mixture studies was investigated in mice immunized with a 1:1:1, 1:2:1, or 1:5:1 mixture of hRSV:hMPV:hPIV3 mRNA or monovalent hMPV mRNA (containing 0.5 µg of pre-fusion hMPV mRNA). Antibody titers induced by these protocols were compared to those induced by a 1:1:1 mixture of hRSV:hMPV:hPIV3 mRNA, and, in terms of hMPV alone, to those induced by monovalent hMPV mRNA.

[0707] For example, through ELISA ( Figure 4A and Figure 4B ) and trace neutralization ( Figure 4D and Figure 4EAs assessed, increasing the hMPV dose by 2 or 5 times had no significant effect on immunogenicity against hRSV or hPIV3. As discussed above, immunization with a 1:1:1 mixture of hRSV:hMPV:hPIV3 mRNA resulted in significantly reduced hMPV neutralizing antibody induction compared to monovalent hMPV mRNA. A 2-fold increase in the hMPV dose (i.e., a 1:2:1 mixture of hRSV:hMPV:hPIV3 mRNA) increased the neutralizing antibody titer to a level similar to that induced by monovalent hMPV mRNA (1.4-fold lower, p = 0.708). Figure 4F Increasing the hMPV dose by 5-fold (i.e., a 1:5:1 mixture of hRSV:hMPV:hPIV3 mRNA) further enhanced immunogenicity, resulting in a 3.7-fold increase in antibody binding titer compared to 0.5 µg monovalent hMPV (p < 0.001). Figure 4C Furthermore, the neutralizing antibody titer was 1.4 times higher (p = 0.835). Figure 4F Data from this mixture study demonstrate that a 5-fold increase in hMPV dose overcomes interference without adversely affecting the immunogenicity of hRSV or hPIV3.

[0708] The effect of increasing hPIV3 dosage in mixture studies was investigated in mice immunized with either a 1:1:0.2 mixture of hRSV:hMPV:hPIV3 mRNA or monovalent hPIV3 mRNA (containing 0.1 µg of hPIV3 mRNA). Antibody titers induced by these regimens were compared to those induced by a 1:1:1 mixture of hRSV:hMPV:hPIV3 mRNA, and additionally, only for hPIV3, to those induced by monovalent hPIV3 mRNA. Data from this mixture study demonstrate that, as measured by ELISA (… Figures 5A-5C ) and trace neutralization ( Figures 5D-5F According to the assessment, reducing the hPIV3 dose by 5-fold did not overcome the interference and had no effect on the immunogenicity against hRSV, hMPV, or hPIV3.

[0709] Data from the above mixture studies demonstrate that: (1) the interference appears to be due to mRNA expression; (2) no significant interference was observed when hRSV, hMPV and hPIV3 recombinant proteins were administered together; (3) increasing the hMPV mRNA dose overcomes the interference without negatively affecting the immunogenicity of hRSV or hPIV3; and (4) decreasing the hPIV3 mRNA dose does not overcome the interference. Example 4: Study of trivalent co-prepared formulation and immunogenicity of hMPV

[0710] The effect of increased hMPV dosage in co-formulation studies was also investigated using the same ratios of hRSV, hMPV, and hPIV3 discussed above. Dosages are shown in Table 2 below. Unless otherwise specified, mRNA constructs were formulated with LNP OF-02. Table 2 - Dosage / Ratio of Co-formulated Pre-fusion hRSV, hMPV, and hPIV3 mRNAs as tested with LNP OF-02

[0711] To evaluate the effect of increased hMPV dosage in the co-formulation study, mice were immunized with either a 1:2:1 or 1:5:1 co-formulation of hRSV:hMPV:hPIV3 mRNA, or monovalent mRNA (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). Antibody titers induced by the co-formulated hRSV:hMPV:hPIV3 mRNA were compared with antibody titers induced by the 1:1:1 co-formulation of hRSV:hMPV:hPIV3 mRNA and antibody titers induced by the monovalent control.

[0712] Compared with immunization using monovalent RSV, immunization with the 1:1:1 co-prepared formulation induced similar hRSV-binding and neutralizing antibody titers. A 2-fold and 5-fold increase in hMVP dose did not significantly affect RSV antibody titers. Figure 6A and Figure 6B Similar results were observed regarding hPIV3 antibody titers, with no significant differences between any groups. Figure 6C and Figure 6D This data demonstrates that no interference with hRSV mRNA or hPIV3 mRNA was observed in the co-prepared formulation, and there were no significant differences in the induction of hRSV and hPIV3 binding antibody titers or neutralizing antibody titers.

[0713] Regarding hMPV, compared with immunization using monovalent hMPV, the 1:1:1 co-prepared formulation induced an immune titer 4.3 times lower (p < 0.001) than the binding antibody titer. Figure 6E The neutralizing antibody titer was 6-fold lower (p < 0.001) and lower (p < 0.001). Figure 6FCompared to the 1:1:1 co-prepared formulation, a two-fold increase in hMPV dose (i.e., a 1:2:1 co-prepared formulation of hRSV:hMPV:hPIV3 mRNA) increased binding antibody titers by 1.75-fold (p = 0.0159) and neutralizing antibody titers by 1.52-fold (p = 0.8), respectively. However, both were still significantly lower than the monovalent control (2.44-fold (p < 0.001) and 3.94-fold (p < 0.001), respectively). Compared to the 1:1:1 co-prepared formulation, a five-fold increase in hMPV dose (i.e., a 1:5:1 co-prepared formulation of hRSV:hMPV:hPIV3 mRNA) increased binding antibody titers by 2.57-fold (p < 0.001) and neutralizing antibody titers by 2.53-fold (p = 0.047), respectively. The difference in binding antibody titer relative to monovalent hMPV was very slight (1.66-fold, p = 0.0389), while the difference in neutralizing antibody titer relative to monovalent hMPV was not statistically significant (2.37-fold, p = 0.079).

[0714] To further evaluate the presence of interference and the effect of increased hMPV dose, the co-prepared formulation was prepared using LNPcKK-E10. The doses used are shown in Table 3 below. Table 3 - Dosage / Ratio of Co-formulated Pre-fusion hRSV, hMPV, and hPIV3 mRNAs as tested with LNP cKK-E10

[0715] To evaluate the effect of increased hMPV dosage in studies using the LNP cKK-E10 co-formulation, mice were immunized with either a 1:2:1 or 1:5:1 co-formulation of hRSV:hMPV:hPIV3 mRNA, or monovalent mRNA (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). Antibody titers induced by the co-formulated hRSV:hMPV:hPIV3 mRNA were then compared to antibody titers induced by the monovalent control.

[0716] Compared with immunization using monovalent RSV, immunizations using co-prepared formulations at ratios of 1:2:1 and 1:5:1 induced similar hRSV-binding antibody titers and neutralizing antibody titers. Figure 7A and Figure 7B Similar results were observed regarding hPIV3 antibody titers, with no significant differences between any groups. Figure 7C and Figure 7DThis data demonstrates that no interference with hRSV mRNA or hPIV3 mRNA was observed in the co-formulation with LNP cKK-E10, and there were no significant differences in the induction of hRSV and hPIV3 binding antibody titers or neutralizing antibody titers.

[0717] Regarding hMPV, compared with immunization using monovalent hMPV, the 1:2:1 co-prepared formulation induced an immune titer 1.9 times lower (p = 0.0027) than that of binding antibodies. Figure 7E The neutralizing antibody titer was 3.9 times lower (p < 0.001) and lower (p < 0.001). Figure 7F Increasing the hMPV dose by 5-fold (i.e., a 1:5:1 co-formulation of hRSV:hMPV:hPIV3 mRNA) increased the binding and neutralizing antibody titers to levels similar to those induced by monovalent hMPV.

[0718] Data from co-formulation studies demonstrate that a 5-fold increase in hMPV dosage overcomes interference without adversely affecting the immunogenicity of hRSV or hPIV3. Furthermore, regarding the 1:5:1 co-formulation, mRNA constructs formulated with LNP OF-02 and LNP cKK-E10 exhibited similar interference patterns. Example 5: Study on the effect of trivalent co-formulations of various LNPs on immunogenicity

[0719] The immunogenicity of trivalent co-formulations of pre-fusion hRSV, hMPV, and hPIV3 prepared with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10 was evaluated. The dose / ratio of pre-fusion hRSV, hMPV, and hPIV3 is shown in Table 4 below. Table 4 - Dosage / ratio of co-formulated pre-fusion hRSV, hMPV, and hPIV3 mRNAs tested with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10

[0720] To evaluate the effect on immunogenicity in co-formulations of pre-fusion hRSV, hMPV, and hPIV3 prepared with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10, mice were immunized with either a 1:1:1 or 1:3:1 co-formulation of hRSV:hMPV:hPIV3 mRNA, or monovalent mRNA (containing 0.5 µg / dose of pre-fusion hRSV mRNA, pre-fusion hPIV3 mRNA, or pre-fusion hMPV mRNA). Mice in groups 1–6 (Table 4) were administered the mRNA constructs prepared with LNP OF-02. Mice in groups 7–11 were administered the mRNA construct formulated with LNP cKK-E10, and mice in groups 12–16 were administered the mRNA construct formulated with LNP GL-HEPES-E3-E12-DS-4-E10. Each mRNA construct was diluted in PBS, except for group 1 (i.e., the trehalose-bridged group), in which the mRNA construct was diluted in trehalose. Antibody titers induced by the co-formulated hRSV:hMPV:hPIV3 mRNA construct tested with each of the three different LNPs were compared with antibody titers induced by a monovalent control.

[0721] Western blot was performed on lysates from cells transfected with the following: co-formulations of hRSV:hMPV:hPIV3 mRNA in 1:1:1 and 1:3:1 ratios prepared with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10, and monovalent hRSV, hMPV, and hPIV3 mRNA, to detect hRSV, hMPV, and hPIV3 protein expression in each formulation. Figures 8A-8C All formulations containing hRSV were positive for high levels of protein expression. Figure 8A All formulations containing hMPV showed positive results for high levels of protein expression. Figure 8B All formulations containing hPIV3 were positive for high levels of protein expression. Figure 8C ).

[0722] Regarding hMPV, compared with immunization using monovalent hMPV, immunization induced by the 1:1:1 co-preparation of LNP OF-02 resulted in 3.9-fold (p < 0.001) lower binding antibody titers and 6.8-fold (p < 0.001) lower neutralizing antibody titers. Figure 9A and 9BA 3-fold increase in hMPV dose tested with LNP OF-02 (i.e., a 1:3:1 co-formulation of hRSV:hMPV:hPIV3 mRNA) increased both binding and neutralizing antibody titers, but both were still lower than those induced by monovalent hMPV control (2-fold lower (p = 0.0145) and 2.2-fold lower (p = 0.0128), respectively). Immunization with the 1:1:1 co-formulation of LNP cKK-E10 induced 2.1-fold (p < 0.008) lower binding antibody titers and 1.9-fold (p < 0.0245) lower neutralizing antibody titers compared to immunization with monovalent hMPV. Figure 9A and 9B A 3-fold increase in the hMPV dose tested with LNP cKK-E10 (i.e., a 1:3:1 co-formulation of hRSV:hMPV:hPIV3 mRNA) increased both binding and neutralizing antibody titers to levels similar to those induced by monovalent hMPV control. Immunization with a 1:1:1 co-formulation of LNP GL-HEPES-E3-E12-DS-4-E10 induced 2-fold lower (p = 0.0147) hMPV binding antibody titers and similar neutralizing antibody titers compared to immunization with monovalent hMPV. Figure 9A and 9B Therefore, no interference with functional anti-hMPV antibody induction was observed. Furthermore, compared to immunization with monovalent hMPV, a 3-fold increase in the hMPV dose tested with LNP GL-HEPES-E3-E12-DS-4-E10 (i.e., a 1:3:1 co-formulation of hRSV:hMPV:hPIV3 mRNA) induced a 1.8-fold (p < 0.0395) higher binding antibody titer and a 4.5-fold (p < 0.001) higher neutralizing antibody titer. Figure 9A and 9B Furthermore, data indicate that monovalent hMPV formulated with LNP OF-02 is more immunogenic than monovalent hMPV formulated with cKK-E10 or GL-HEPES-E3-E12-DS-4-E10.

[0723] Regarding hPIV3, compared with immunization using monovalent hPIV3, immunization using 1:1:1 and 1:3:1 co-preparations of LNP cKK-E10 or LNP GL-HEPES-E3-E12-DS-4-E10 induced similar hPIV3 binding antibody titers and neutralizing antibody titers. Figure 9C and 9DNo interference with hPIV3 immunogenicity was observed in co-formulations using LNP cKK-E10 and LNP GL-HEPES-E3-E12-DS-4-E10. Furthermore, there were no significant differences in the induction of hPIV3-binding or neutralizing antibody titers in either LNP. Immunization with the 1:1:1 and 1:3:1 co-formulations of LNP OF-02 induced similar hPIV3-binding and neutralizing antibody titers compared to immunization with monovalent hPIV3.

[0724] Regarding RSV, compared with the 1:1:1 co-formulation using LNP cKK-E10, the monovalent RSV formulated with LNP cKK-E10 induced a 1.9-fold (p < 0.0316) lower binding antibody titer and a 1.8-fold (not statistically significant) lower neutralizing antibody titer. Figure 9E and 9F Furthermore, monovalent RSV formulated with LNP GL-HEPES-E3-E12-DS-4-E10 exhibited lower immunogenicity than expected. No interference with hRSV was observed in co-formulations with LNP OF-02, LNP cKK-E10, or LNP GL-HEPES-E3-E12-DS-4-E10 at 1:1:1 and 1:3:1 ratios.

[0725] Furthermore, to determine whether PBS had any effect on immunogenicity, the neutralizing antibody titers of hRSV, hMPV, and hPIV3 mRNA constructs diluted with PBS were compared with those diluted with trehalose. The results showed that the mRNA constructs diluted with PBS induced similar neutralizing antibody titers of hRSV, hMPV, and hPIV3 compared to those diluted with trehalose. Figure 10 Therefore, the mRNA construct diluted in PBS had no effect on immunogenicity compared to the mRNA construct diluted in trehalose.

[0726] Data from the above studies of trivalent co-formulations using LNP OF-02, LNP cKK-E10, and LNP GL-HEPES-E3-E12-DS-4-E10 demonstrate that: (1) different LNPs have different effects on mRNA expression, which may lead to interference; (2) no interference with hMPV immunogenicity was observed in the co-formulation using LNP GL-HEPES-E3-E12-DS-4-E10; (3) slight interference with hMPV immunogenicity was observed in the 1:1:1 co-formulation using LNP cKK-E10, but no interference was observed in the 1:3:1 co-formulation using LNP cKK-E10; (4) no interference was observed in the co-formulation using LNP OF-02, LNP cKK-E10, or LNP... No interference with the immunogenicity of hRSV or hPIV3 was observed in the co-formulation of GL-HEPES-E3-E12-DS-4-E10; and (5) switching from trehalose to PBS as a diluent did not affect the immunogenicity. Example 6: Study on the effect of bivalent co-formulations of various LNPs on immunogenicity

[0727] The interference of pre-fusion hRSV and hMPV 1:1 co-formulations prepared with LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP OF-02 on the immunogenicity of hRSV and hMPV was evaluated. Furthermore, the immunogenicity of hRSV and hMPV in the 1:1 co-formulations tested with IM-001 was compared with that tested with GL-HEPES-E3-E12-DS-4-E10 and OF-02. The dose / ratio of pre-fusion hRSV and hMPV is shown in Table 5 below. Table 5 - Dosage / ratio of co-formulated pre-fusion hRSV and hMPV tested with LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or OF-02

[0728] To evaluate hRSV and hMPV interference and immunogenicity in co-formulations of pre-fusion hRSV and hMPV prepared with LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP OF-02, mice were immunized with either a 1:1 co-formulation of hRSV:hMPV mRNA or monovalent mRNA (containing 0.5 µg / dose of pre-fusion hRSV mRNA or pre-fusion hPIV3 mRNA). Mice in groups 1–3 (Table 5) were administered the 1:1 co-formulation of hRSV:hMPV mRNA prepared with LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP OF-02. Mice in groups 4–6 were administered monovalent hRSV mRNA of LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP OF-02, and mice in groups 7–9 were administered monovalent hMPV mRNA of LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP OF-02. Antibody titers induced by the co-formulated hRSV:hMPV mRNA constructs tested with each of the three different LNPs were compared with each other and with antibody titers induced by the monovalent control.

[0729] No interference with hRSV immunogenicity was observed in any of the bivalent 1:1 co-prepared formulations of pre-fusion hRSV and hMPV formulated with LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, or LNP OF-02. Figure 11A and 11B Furthermore, no interference with hMPV immunogenicity was observed in the bivalent 1:1 co-prepared formulations of pre-fusion hRSV and hMPV formulated with LNP IM-001 and LNP GL-HEPES-E3-E12-DS-4-E10. Figure 11C and 11D However, compared with immunization using monovalent hMPV, the bivalent 1:1 co-preparation tested with OF-02 induced a 1.9-fold (p < 0.0173) lower (binding antibody titer) and a 2.2-fold (not statistically significant) lower (neutralizing antibody titer) lower (binding antibody titer). Figure 11C and 11D ).

[0730] Regarding hRSV immunogenicity, compared with the bivalent 1:1 co-formulation tested with LNP IM-001, the bivalent 1:1 co-formulation was more immunogenic and induced a 2.4-fold higher RSV neutralizing antibody titer. Figure 11A Furthermore, compared to immunization with the bivalent 1:1 co-prepared formulation tested with LNP OF-02, the bivalent 1:1 co-prepared formulation tested with LNP IM-001 induced similar hRSV binding antibody titers and neutralizing antibody titers. Figure 11A and 11B Furthermore, the bivalent 1:1 co-formulation tested with LNP IM-001 was more immunogenic than the monovalent hRSV, inducing 2.4-fold (p = 0.0047) higher neutralizing antibody titers and 1.7-fold (p = 0.0037) higher binding antibody titers.

[0731] Regarding hMPV immunogenicity, compared with the bivalent 1:1 co-formulation tested with LNP IM-001, the bivalent 1:1 co-formulation was more immunogenic and induced a 2.8-fold (p = 0.0211) higher hMPV neutralizing antibody titer. Figure 11C Furthermore, compared to immunization with the bivalent 1:1 co-prepared formulation tested with LNP OF-02, the bivalent 1:1 co-prepared formulation tested with LNP IM-001 induced similar hMPV binding antibody titers and neutralizing antibody titers. Figure 11C and 11D Furthermore, the bivalent 1:1 co-formulation tested with LNP IM-001 was more immunogenic than the monovalent hMPV, inducing a 3.2-fold (p < 0.0093) higher neutralizing antibody titer, while the bivalent 1:1 co-formulation tested with LNP GL-HEPES-E3-E12-DS-4-E10 induced a 9.5-fold (p < 0.001) higher neutralizing antibody titer than the monovalent hMPV tested with LNP GL-HEPES-E3-E12-DS-4-E10.

[0732] Data from the above studies of bivalent co-formulations using LNP IM-001, LNP GL-HEPES-E3-E12-DS-4-E10, and LNP OF-02 demonstrate that: (1) no interference with the immunogenicity of hRSV or hMPV was observed in the bivalent co-formulations tested with LNP IM-001 or LNP GL-HEPES-E3-E12-DS-4-E10; (2) the bivalent co-formulations tested with IM-001 were more immunogenic than those tested with LNP GL-HEPES-E3-E12-DS-4-E10; and (3) the antibody titers in the 1:1 co-formulations were generally higher than those in the monovalent controls. Example 7: Compatibility of bivalent co-formulations of RSV and hMPV when applied with hPIV3 mixture

[0733] The interference of pre-fusion hRSV and hMPV bivalent co-formulations with the hPIV3 mixture on the immunogenicity of hRSV, hMPV, and hPIV3 was evaluated. The dosages of pre-fusion hRSV, hMPV, and hPIV3 are shown in Table 6 below. Table 6 - Dosage of pre-fusion hRSV and hMPV and pre-fusion hPIV3 in bivalent co-formulations

[0734] To evaluate the effect of the bivalent co-formulation of pre-fusion hRSV and hMPV administered with hPIV3 mixture on the immunogenicity of hRSV, hMPV, and hPIV3, mice were immunized with the following groups: a 1:1 bivalent hRSV:hMPV co-formulation, a 1:1 bivalent hRSV:hMPV co-formulation administered with hPIV3 (containing 0.5 µg / dose of pre-fusion hPIV3 mRNA), or monovalent hPIV3 (containing 0.5 µg / dose of pre-fusion hPIV3 mRNA) as a control. Each mRNA construct was formulated with LNP GL-HEPES-E3-E12-DS-4-E10 and diluted in PBS. The antibody titers induced by the bivalent 1:1 co-preparation of hRSV:hMPV administered with hPIV3 were compared with the antibody titers induced by the bivalent 1:1 co-preparation of hRSV:hMPV and the antibody titers induced by the monovalent hPIV3 control.

[0735] Compared with immunizations using a bivalent 1:1 co-preparation of hRSV:hMPV alone and a monovalent hPIV3 control alone, immunizations using a bivalent 1:1 co-preparation of hRSV:hMPV along with a mixture of hPIV3 induced similar hRSV, hMPV, and hPIV3 binding antibody titers and neutralizing antibody titers. Figure 12A and Figure 12B No statistically significant differences were observed in the titers of hRSV, hMPV, and hPIV3 binding or neutralizing antibodies. This data demonstrate that the administration of a bivalent co-formulation of hRSV:hMPV with an hPIV3 mixture in the LNP GL-HEPES-E3-E12-DS-4-E10 formulation does not interfere with the immunogenicity of hRSV, hMPV, or hPIV3.

Claims

1. A composition comprising at least two messenger RNAs (mRNAs), wherein the at least two mRNAs comprise an open reading frame (ORF) encoding a recombinant F protein antigenic polypeptide, selected from the group consisting of: (i) The first mRNA encoding the human respiratory syncytial virus (hRSV) F protein antigen containing the amino acid sequence of SEQ ID NO: 1; (ii) A second mRNA encoding the human metapneumovirus (hMPV) F protein antigen containing the amino acid sequence of SEQ ID NO: 2; and (iii) A third mRNA encoding the human parainfluenza virus 3 (hPIV3) F protein antigen containing the amino acid sequence of SEQ ID NO:

3.

2. The composition of claim 1, comprising: The first mRNA encoding the amino acid sequence of SEQ ID NO: 1; and The second mRNA encoding the amino acid sequence of SEQ ID NO:

2.

3. The composition of claim 1, comprising: The first mRNA encoding the amino acid sequence of SEQ ID NO: 1; and The third mRNA encoding the amino acid sequence of SEQ ID NO:

3.

4. The composition of claim 1, comprising: The second mRNA encoding the amino acid sequence of SEQ ID NO: 2; and The third mRNA encoding the amino acid sequence of SEQ ID NO:

3.

5. The composition of claim 1, comprising at least three mRNAs, wherein the at least three mRNAs include: The first mRNA encoding the amino acid sequence of SEQ ID NO: 1; This is the second mRNA encoding the amino acid sequence of SEQ ID NO: 2; as well as The third mRNA encoding the amino acid sequence of SEQ ID NO:

3.

6. The composition of claim 1, comprising three mRNAs, the three mRNAs comprising: The first mRNA encoding the amino acid sequence of SEQ ID NO: 1; This is the second mRNA encoding the amino acid sequence of SEQ ID NO: 2; as well as The third mRNA encoding the amino acid sequence of SEQ ID NO:

3.

7. The composition according to any one of claims 1-3, 5 or 6, wherein the hRSV F protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

1.

8. The composition of any one of claims 1, 2 or 4-6, wherein the hMPV F protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

2.

9. The composition of any one of claims 1 or 3-6, wherein the hPIV3 F protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

3.

10. The composition of any one of claims 1-3, 5 or 6, wherein the first mRNA comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO:

4.

11. The composition of any one of claims 1, 2 or 4-6, wherein the second mRNA comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO:

5.

12. The composition of any one of claims 1 or 3-6, wherein the third mRNA comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO:

6.

13. The composition as claimed in any of the preceding claims, wherein at least one of these recombinant F protein antigenic polypeptides is a pre-fusion protein.

14. The composition as claimed in any of the preceding claims, wherein at least one of the mRNAs comprises a codon-optimized ORF.

15. The composition of any one of the preceding claims, wherein at least one of the mRNAs comprises at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and at least one polyadenylated (poly(A)) sequence.

16. The composition of any one of the preceding claims, wherein at least one of the mRNAs comprises a 5' UTR containing a nucleic acid sequence having at least 80% identity with SEQ ID NO:

7.

17. The composition as claimed in any of the preceding claims, wherein at least one of these mRNAs comprises a 3' UTR containing a nucleic acid sequence having at least 80% identity with SEQ ID NO:

8.

18. The composition as claimed in any of the preceding claims, wherein at least one of the mRNAs comprises at least one chemical modification.

19. The composition of any one of claims 1 to 18, wherein 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 at least one of these mRNAs are chemically modified.

20. The composition of any one of claims 1 to 18, wherein 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 at least one of these ORFs are chemically modified.

21. The composition of any one of claims 18-20, wherein the chemical modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-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-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

22. The composition of claim 21, wherein the chemical modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

23. The composition of claim 22, wherein the chemical modification is N1-methylpseuuridine.

24. The composition of any one of claims 1-23, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:1:1 (w / w).

25. The composition of any one of claims 1-23, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:2:1 (w / w).

26. The composition of any one of claims 1-23, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:3:1 (w / w).

27. The composition of any one of claims 1-23, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:5:1 (w / w).

28. The composition of any one of claims 1-23, wherein the first mRNA, the second mRNA and the third mRNA are present in a ratio (w / w) of about 1:1:1 to about 1:10:

1.

29. The composition of any one of claims 1-23, wherein the first mRNA, the second mRNA and the third mRNA are present in a ratio (w / w) of about 1:1:0.1 to about 1:1:

1.

30. The composition of any one of claims 24 to 29, wherein the ratio is expressed in micrograms (µg).

31. The composition of claim 24, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 1 microgram of the second mRNA to about 1 microgram of the third mRNA.

32. The composition of claim 25, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 2 micrograms of the second mRNA to about 1 microgram of the third mRNA.

33. The composition of claim 26, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 3 micrograms of the second mRNA to about 1 microgram of the third mRNA.

34. The composition of claim 27, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 5 micrograms of the second mRNA to about 1 microgram of the third mRNA.

35. The composition of claim 24, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

36. The composition of claim 26, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

37. The composition of claim 29, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.1 micrograms of the third mRNA.

38. The composition of claim 25, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1 microgram of the second mRNA to about 0.5 micrograms of the third mRNA.

39. The composition of claim 27, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 2.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

40. The composition of any of the preceding claims, wherein the first mRNA, the second mRNA, and the third mRNA are not covalently linked to each other.

41. The composition of any one of claims 1-39, wherein one or more of the first mRNA, the second mRNA, and the third mRNA are covalently linked to each other.

42. The composition of any one of claims 1-41, wherein the first mRNA, the second mRNA and the third mRNA are each formulated into individual nanoparticles (LNPs).

43. The composition according to any one of claims 1-41, wherein the first mRNA, the second mRNA, and the third mRNA are formulated into the same LNP.

44. The composition of claim 42 or 43, wherein the LNP comprises at least one cationic lipid.

45. The composition of claim 44, wherein the cationic lipid is biodegradable.

46. ​​The composition of claim 44, wherein the cationic lipid is not biodegradable.

47. The composition of claim 44, wherein the cationic lipid is cleavable.

48. The composition of claim 44, wherein the cationic lipid is not cleavable.

49. The composition of claim 44, wherein 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, GL-HEPES-E3-E12-DS-3-E14 and IM-001.

50. The composition of claim 49, wherein the cationic lipid is cKK-E10.

51. The composition of claim 49, wherein the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

52. The composition of claim 49, wherein the cationic lipid is IM-001.

53. The composition of any one of claims 42-52, wherein the LNP further comprises polyethylene glycol (PEG) conjugated (PEGylated) lipids, cholesterol-based lipids, and auxiliary lipids.

54. The composition of any one of claims 42-53, wherein the LNP comprises: Cationic lipids with a molar ratio of 35% to 55%; A polyethylene glycol (PEG) conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids with a molar ratio of 20% to 45%; and The molar ratio of auxiliary lipids is 5% to 35%. All of these molar ratios are relative to the total lipid content of the LNP.

55. The composition of claim 54, wherein the LNP comprises: Cationic lipids with a molar ratio of 40%; PEGylated lipids with a molar ratio of 1.5%; Cholesterol-based lipids with a molar ratio of 28.5%; and The molar ratio of auxiliary lipids is 30%.

56. The composition of any one of claims 53-55, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

57. The composition of any one of claims 53-56, wherein the cholesterol-based lipid is cholesterol.

58. The composition of any one of claims 53-57, wherein the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

59. The composition of any one of claims 42-58, wherein the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

60. The composition of any one of claims 42-58, wherein the LNP comprises: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

61. The composition of any one of claims 42-58, wherein the LNP comprises: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

62. The composition according to any one of claims 42-61, wherein the average diameter of the LNP is 30 nm to 200 nm.

63. The composition of claim 62, wherein the average diameter of the LNP is 80 nm to 150 nm.

64. The composition according to any one of claims 1-63, wherein at least one of these mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ； (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Clustered (A) tail.

65. A composition comprising at least two messenger RNAs (mRNAs), wherein each of the at least two mRNAs comprises an open reading frame (ORF) encoding an antigenic polypeptide of a recombinant F protein, wherein at least one of the mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ； (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

66. A composition comprising at least two messenger RNAs (mRNAs), wherein each of the at least two mRNAs comprises an open reading frame (ORF) encoding an antigenic polypeptide of a recombinant F protein, wherein at least one of the mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ； (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

67. A composition comprising at least two messenger RNAs (mRNAs), wherein each of the at least two mRNAs comprises an open reading frame (ORF) encoding an antigenic polypeptide of a recombinant F protein, wherein at least one of the mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ； (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs), which contain: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

68. A composition comprising at least two messenger RNAs (mRNAs), wherein each of the at least two mRNAs comprises an open reading frame (ORF) encoding an antigenic polypeptide of a recombinant F protein, wherein at least one of the mRNAs comprises the following structural elements: (i) A 5' cap with the following structure: ； (ii) The 5' untranslated region (5' UTR) of the nucleic acid sequence of SEQ ID NO: 7; (iii) A protein-coding region having a nucleic acid sequence of SEQ ID NO: 4, 5 or 6; (iv) The 3' untranslated region (3' UTR) of the nucleic acid sequence of SEQ ID NO: 8; and (v) Acrylative tails; The mRNA is formulated in lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10, IM-001, OF-02 or cKK-E10 in a molar ratio of 35% to 55%.

69. A method for inducing an immune response to hRSV or protecting a subject from hRSV infection, the method comprising administering to the subject the composition as described in any one of claims 1-68.

70. A method for inducing an immune response to hMPV or protecting a subject from hMPV infection, the method comprising administering to the subject the composition as described in any one of claims 1-68.

71. A method for inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the method comprising administering to the subject the composition as described in any one of claims 1-68.

72. The method of claim 69, wherein, after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hRSV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO:

1.

73. The method of claim 70, wherein after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hMPV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO:

2.

74. The method of claim 71, wherein after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hPIV3 neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hPIV3 F protein antigen of SEQ ID NO:

3.

75. The method of claim 69, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies relative to a subject administered a protein hRSV vaccine.

76. The method of claim 70, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies relative to a subject administered the protein hMPV vaccine.

77. The method of claim 71, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hPIV3 neutralizing antibodies relative to a subject administered the protein hPIV3 vaccine.

78. The method of claim 69, wherein the composition increases the serum concentration of neutralizing antibodies in subjects pre-immunized with hRSV.

79. The method of claim 70, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV.

80. The method of claim 71, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hPIV3.

81. A composition for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering the composition as described in any one of claims 1-68 to the subject.

82. A composition for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering the composition as described in any one of claims 1-68 to the subject.

83. A composition for use in inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the use comprising administering to the subject the composition as described in any one of claims 1-68.

84. Use of the composition of any one of claims 1-68 in the manufacture of a medicament for inducing an immune response to hRSV or for protecting a subject from hRSV infection.

85. Use of the composition of any one of claims 1-68 in the manufacture of a medicament for inducing an immune response to hMPV or for protecting a subject from hMPV infection.

86. Use of the composition of any one of claims 1-68 in the manufacture of a medicament for inducing an immune response to hPIV3 or for protecting a subject from hPIV3 infection.

87. A composition comprising three messenger RNAs (mRNAs), wherein: (i) The first mRNA encodes the human respiratory syncytial virus (hRSV) F protein antigen containing the amino acid sequence of SEQ ID NO: 1; (ii) The second mRNA encodes the human metapneumovirus (hMPV) F protein antigen containing the amino acid sequence of SEQ ID NO: 2; and (iii) The third mRNA encodes the human parainfluenza virus 3 (hPIV3) F protein antigen containing the amino acid sequence of SEQ ID NO: 3; The first mRNA, the second mRNA, and the third mRNA are formulated into the same lipid nanoparticle (LNP), which contains: OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14 or IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

88. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:1:1 (w / w).

89. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:2:1 (w / w).

90. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:3:1 (w / w).

91. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:5:1 (w / w).

92. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio (w / w) of about 1:1:1 to about 1:10:

1.

93. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio (w / w) of about 1:1:0.1 to about 1:1:

1.

94. The composition of any one of claims 88-93, wherein the ratio is expressed in micrograms (µg).

95. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio (w / w) of about 1 microgram of the first mRNA to about 1 microgram of the second mRNA to about 1 microgram of the third mRNA.

96. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 2 micrograms of the second mRNA to about 1 microgram of the third mRNA.

97. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 3 micrograms of the second mRNA to about 1 microgram of the third mRNA.

98. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 5 micrograms of the second mRNA to about 1 microgram of the third mRNA.

99. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

100. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

101. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.1 micrograms of the third mRNA.

102. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1 microgram of the second mRNA to about 0.5 micrograms of the third mRNA.

103. The composition of claim 87, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 2.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

104. A method for inducing an immune response to hRSV or protecting a subject from hRSV infection, the method comprising administering to the subject the composition as described in any one of claims 87-103.

105. A method for inducing an immune response to hMPV or protecting a subject from hMPV infection, the method comprising administering to the subject the composition as described in any one of claims 87-103.

106. A method for inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the method comprising administering to the subject the composition as described in any one of claims 87-103.

107. The method of claim 104, wherein, after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hRSV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO:

1.

108. The method of claim 105, wherein, after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hMPV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO:

2.

109. The method of claim 106, wherein after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hPIV3 neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hPIV3 F protein antigen of SEQ ID NO:

3.

110. The method of claim 104, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies relative to a subject administered a protein hRSV vaccine.

111. The method of claim 105, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies relative to a subject administered the protein hMPV vaccine.

112. The method of claim 106, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hPIV3 neutralizing antibodies relative to a subject administered the protein hPIV3 vaccine.

113. The method of claim 104, wherein the composition increases the serum concentration of neutralizing antibodies in subjects pre-immunized with hRSV.

114. The method of claim 105, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV.

115. The method of claim 106, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hPIV3.

116. A composition for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering to the subject the composition as described in any one of claims 87-103.

117. A composition for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering to the subject the composition as described in any one of claims 87-103.

118. A composition for use in inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the use comprising administering to the subject the composition as described in any one of claims 87-103.

119. Use of the composition of any one of claims 87-103 in the manufacture of a medicament for inducing an immune response to hRSV or for protecting a subject from hRSV infection.

120. Use of the composition of any one of claims 87-103 in the manufacture of a medicament for inducing an immune response to hMPV or protecting a subject from hMPV infection.

121. Use of the composition of any one of claims 87-103 in the manufacture of a medicament for inducing an immune response to hMPV or for protecting a subject from hPIV3 infection.

122. A composition comprising three messenger RNAs (mRNAs), wherein: (i) The first mRNA encodes the human respiratory syncytial virus (hRSV) F protein antigen containing the amino acid sequence of SEQ ID NO: 1; (ii) The second mRNA encodes the human metapneumovirus (hMPV) F protein antigen containing the amino acid sequence of SEQ ID NO: 2; and (iii) The third mRNA encodes the human parainfluenza virus 3 (hPIV3) F protein antigen containing the amino acid sequence of SEQ ID NO:

3.

123. The composition of claim 122, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:1:1 (w / w).

124. The composition of claim 122, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:2:1 (w / w).

125. The composition of claim 122, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:3:1 (w / w).

126. The composition of claim 122, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of about 1:5:1 (w / w).

127. The composition of claim 122, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio (w / w) of about 1:1:1 to about 1:10:

1.

128. The composition of claim 122, wherein the first mRNA, the second mRNA and the third mRNA are present in a ratio (w / w) of about 1:1:0.1 to about 1:1:

1.

129. The composition of any one of claims 123-128, wherein the ratio is expressed in micrograms (µg).

130. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 1 microgram of the second mRNA to about 1 microgram of the third mRNA.

131. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 2 micrograms of the second mRNA to about 1 microgram of the third mRNA.

132. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 3 micrograms of the second mRNA to about 1 microgram of the third mRNA.

133. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 5 micrograms of the second mRNA to about 1 microgram of the third mRNA.

134. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

135. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

136. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA to about 0.1 micrograms of the third mRNA.

137. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1 microgram of the second mRNA to about 0.5 micrograms of the third mRNA.

138. The composition of claim 129, wherein the first mRNA, the second mRNA, and the third mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 2.5 micrograms of the second mRNA to about 0.5 micrograms of the third mRNA.

139. A method for inducing an immune response to hRSV or protecting a subject from hRSV infection, the method comprising administering to the subject the composition as described in any one of claims 122-138.

140. A method for inducing an immune response to hMPV or protecting a subject from hMPV infection, the method comprising administering to the subject the composition as described in any one of claims 122-138.

141. A method for inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the method comprising administering to the subject the composition as described in any one of claims 122-138.

142. The method of claim 139, wherein, after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hRSV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO:

1.

143. The method of claim 140, wherein after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hMPV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO:

2.

144. The method of claim 141, wherein after administration of the composition, the subject has substantially the same or higher serum concentration of anti-hPIV3 neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hPIV3 F protein antigen of SEQ ID NO:

3.

145. The method of claim 139, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies relative to a subject administered a protein hRSV vaccine.

146. The method of claim 140, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies relative to a subject administered the protein hMPV vaccine.

147. The method of claim 141, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hPIV3 neutralizing antibodies relative to a subject administered the protein hPIV3 vaccine.

148. The method of claim 139, wherein the composition increases the serum concentration of neutralizing antibodies in subjects pre-immunized with hRSV.

149. The method of claim 140, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV.

150. The method of claim 141, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hPIV3.

151. A composition for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering to the subject the composition as described in any one of claims 122-138.

152. A composition for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering to the subject the composition as described in any one of claims 122-138.

153. A composition for use in inducing an immune response to hPIV3 or protecting a subject from hPIV3 infection, the use comprising administering to the subject the composition as described in any one of claims 122-138.

154. Use of the composition of any one of claims 122-138 in the manufacture of a medicament for inducing an immune response to hRSV or for protecting a subject from hRSV infection.

155. Use of the composition of any one of claims 122-138 in the manufacture of a medicament for inducing an immune response to hMPV or for protecting a subject from hMPV infection.

156. Use of the composition of any one of claims 122-138 in the manufacture of a medicament for inducing an immune response to hMPV or for protecting a subject from hPIV3 infection.

157. A composition comprising a first messenger RNA (mRNA) and a second mRNA, wherein: (i) The first mRNA encodes the human respiratory syncytial virus (hRSV) F protein antigen containing the amino acid sequence of SEQ ID NO: 1; and (ii) The second mRNA encodes the human metapneumovirus (hMPV) F protein antigen containing the amino acid sequence of SEQ ID NO:

2.

158. The composition of claim 157, wherein the first mRNA and the second mRNA are present in a ratio of about 1:1 (w / w).

159. The composition of claim 157, wherein the first mRNA and the second mRNA are present in a ratio of about 1:2 (w / w).

160. The composition of claim 157, wherein the first mRNA and the second mRNA are present in a ratio of about 1:3 (w / w).

161. The composition of claim 157, wherein the first mRNA and the second mRNA are present in a ratio of about 1:5 (w / w).

162. The composition of claim 157, wherein the first mRNA and the second mRNA are present in a ratio of about 1:1 to about 1:10 (w / w).

163. The composition of any one of claims 157-162, wherein the ratio is expressed in micrograms (µg).

164. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 0.5 micrograms of the second mRNA.

165. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1.5 micrograms of the second mRNA.

166. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 1 microgram of the second mRNA.

167. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 0.5 micrograms of the first mRNA to about 2.5 micrograms of the second mRNA.

168. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 1 microgram of the second mRNA.

169. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 2 micrograms of the second mRNA.

170. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 3 micrograms of the second mRNA.

171. The composition of claim 157, wherein the first mRNA and the second mRNA are present at a ratio (w / w) of about 1 microgram of the first mRNA to about 5 micrograms of the second mRNA.

172. The composition of any one of claims 157-171, wherein the first mRNA and the second mRNA are each formulated into separate lipid nanoparticles (LNPs).

173. The composition of any one of claims 157-171, wherein the first mRNA and the second mRNA are formulated into the same LNP.

174. The composition of claim 172 or 173, wherein the LNP comprises at least one cationic lipid.

175. The composition of claim 174, wherein the cationic lipid is biodegradable.

176. The composition of claim 174, wherein the cationic lipid is not biodegradable.

177. The composition of claim 174, wherein the cationic lipid is cleavable.

178. The composition of claim 174, wherein the cationic lipid is not cleavable.

179. The composition of claim 174, wherein 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, GL-HEPES-E3-E12-DS-3-E14 and IM-001.

180. The composition of claim 179, wherein the cationic lipid is cKK-E10.

181. The composition of claim 179, wherein the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

182. The composition of claim 179, wherein the cationic lipid is IM-001.

183. The composition of any one of claims 174-182, wherein the LNP further comprises polyethylene glycol (PEG) conjugated (PEGylated) lipids, cholesterol-based lipids, and auxiliary lipids.

184. The composition of any one of claims 174-183, wherein the LNP comprises: Cationic lipids with a molar ratio of 35% to 55%; A polyethylene glycol (PEG) conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids with a molar ratio of 20% to 45%; and The molar ratio of auxiliary lipids is 5% to 35%. All of these molar ratios are relative to the total lipid content of the LNP.

185. The composition of any one of claims 174-184, wherein the LNP comprises: Cationic lipids with a molar ratio of 40%; PEGylated lipids with a molar ratio of 1.5%; Cholesterol-based lipids with a molar ratio of 28.5%; and The molar ratio of auxiliary lipids is 30%.

186. The composition of any one of claims 183-185, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

187. The composition of any one of claims 183-186, wherein the cholesterol-based lipid is cholesterol.

188. The composition of any one of claims 183-187, wherein the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

189. The composition of any one of claims 174-188, wherein the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

190. The composition of any one of claims 174-188, wherein the LNP comprises: cKK-E10 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

191. The composition of any one of claims 174-188, wherein the LNP comprises: IM-001 with a molar ratio of 40%; DMG-PEG2000 with a molar ratio of 1.5%; Cholesterol with a molar ratio of 28.5%; and DOPE with a molar ratio of 30%.

192. The composition of any one of claims 174-191, wherein the average diameter of the LNP is 30 nm to 200 nm.

193. The composition according to any one of claims 174-191, wherein the average diameter of the LNP is 80 nm to 150 nm.

194. A method for inducing an immune response to hRSV or protecting a subject from hRSV infection, the method comprising administering to the subject the composition as described in any one of claims 157-193.

195. A method for inducing an immune response to hMPV or protecting a subject from hMPV infection, the method comprising administering to the subject the composition as described in any one of claims 157-193.

196. The method of claim 194, wherein, after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hRSV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hRSV F protein antigen of SEQ ID NO:

1.

197. The method of claim 195, wherein, after administration of the composition, the subject has a substantially the same or higher serum concentration of anti-hMPV neutralizing antibody compared to a subject administered a single antigenic composition comprising an mRNA ORF encoding the hMPV F protein antigen of SEQ ID NO:

2.

198. The method of claim 194, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hRSV neutralizing antibodies relative to a subject administered a protein hRSV vaccine.

199. The method of claim 195, wherein after administration of the composition, the subject has a comparable serum concentration of anti-hMPV neutralizing antibodies relative to a subject administered the protein hMPV vaccine.

200. The method of claim 194, wherein the composition increases the serum concentration of neutralizing antibodies in subjects pre-immunized with hRSV.

201. The method of claim 195, wherein the composition increases the serum concentration of neutralizing antibodies in subjects immunized with pre-existing hMPV.

202. A composition for use in inducing an immune response to hRSV or protecting a subject from hRSV infection, the use comprising administering to the subject the composition as described in any one of claims 157-193.

203. A composition for use in inducing an immune response to hMPV or protecting a subject from hMPV infection, the use comprising administering to the subject the composition as described in any one of claims 157-193.

204. Use of the composition of any one of claims 157-193 in the manufacture of a medicament for inducing an immune response to hRSV or for protecting a subject from hRSV infection.

205. Use of the composition of any one of claims 157-193 in the manufacture of a medicament for inducing an immune response to hMPV or for protecting a subject from hMPV infection.

Images