Polyvalent influenza mRNA vaccine

Abstract

This disclosure provides a polyvalent influenza vaccine composition comprising at least three messenger RNAs (mRNAs) encoding a combination of hemagglutinin (HA) antigens of influenza A and influenza B, wherein the mRNA encoding the HA antigen of influenza A virus is present in a different ratio (w / w) than the mRNA encoding the influenza B virus, and a method for inducing an immune response by administering the composition. In particular, this disclosure relates to mRNA encoding these antigens incorporated into lipid nanoparticles (LNPs).

Description

Technical Field

[0001] Related Applications This application claims priority to European Patent Application No. 23315259.4, filed on June 28, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Messenger RNA (mRNA)-based vaccines are promising alternatives to conventional subunit vaccines that contain antigenic proteins derived from pathogens. Antigenic proteins are usually produced by recombinant gene technology and require bacterial fermentation and / or cell culture, as well as complex purification. mRNA-based vaccines enable the novel expression of complex antigens in vaccinated subjects, thereby allowing for proper post-translational modification and presentation of the antigens in their native conformation. Different from the prior art, the production of mRNA vaccines does not require complex and costly bacterial fermentation, tissue culture, and purification processes. Furthermore, once established, the production process for mRNA vaccines can be used for various antigens, enabling the rapid development and deployment of mRNA vaccines. Moreover, mRNA vaccines are essentially safe delivery vectors as they only transiently express antigens and do not integrate into the host genome. The antigens encoded by mRNA are produced in vivo in vaccinated individuals, so mRNA vaccines are particularly effective in inducing both humoral immunity and T cell-mediated immunity.

[0003] Current mRNA-based polyvalent influenza vaccines under investigation encode hemagglutinin (HA) antigens of both influenza A and influenza B viruses in equal proportions (w / w). While these vaccines are immunogenic, ongoing trials have shown that the immunogenicity of strain B is lower than that of strain A, and that the human immune response to influenza B virus is not optimal with these vaccines. Therefore, there is a need for an RNA-based polyvalent influenza vaccine with improved immunogenicity of strain B. [Overview of the Initiative] [Means for solving the problem]

[0004] In one embodiment, the present disclosure provides a composition comprising at least three messenger RNAs (mRNAs), the at least three mRNAs comprising an open reading frame (ORF) encoding an HA antigen selected from the group consisting of (i) a first mRNA encoding the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encoding the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encoding the HA antigen of a first influenza B virus, wherein the mRNA encoding the HA antigen of the influenza A virus is present in a different ratio (w / w) to the mRNA encoding the HA antigen of the influenza B virus.

[0005] In certain embodiments, the composition comprises a fourth mRNA encoding the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are from different strains).

[0006] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 (w / w).

[0007] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:3 (w / w).

[0008] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:4 (w / w).

[0009] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:5 (w / w).

[0010] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:6 (w / w).

[0011] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:7 (w / w).

[0012] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:8 (w / w).

[0013] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:9 (w / w).

[0014] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:10 (w / w).

[0015] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w).

[0016] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:2:2 (w / w).

[0017] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:3:3 (w / w).

[0018] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:4:4 (w / w).

[0019] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:5:5 (w / w).

[0020] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:6:6 (w / w).

[0021] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:7:7 (w / w).

[0022] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:8:8 (w / w).

[0023] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:9:9 (w / w).

[0024] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:10:10 (w / w).

[0025] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA present in a ratio (w / w) of from about 1:1:2:2 to about 1:1:10:10.

[0026] In certain embodiments, the ratio is expressed in micrograms (μg).

[0027] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA present in a ratio (w / w) of about 32 micrograms of the first mRNA to about 32 micrograms of the second mRNA to about 64 micrograms of the third mRNA to about 64 micrograms of the fourth mRNA.

[0028] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA present in a ratio (w / w) of about 16 micrograms of the first mRNA to about 16 micrograms of the second mRNA to about 64 micrograms of the third mRNA to about 64 micrograms of the fourth mRNA.

[0029] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA present in a ratio (w / w) of about 16 micrograms of the first mRNA to about 16 micrograms of the second mRNA to about 96 micrograms of the third mRNA to about 96 micrograms of the fourth mRNA.

[0030] In certain embodiments, the composition comprises a total of 130 micrograms of mRNA.

[0031] In certain embodiments, the composition comprises a total of 160 micrograms of mRNA.

[0032] In certain embodiments, the composition comprises a total of 200 micrograms of mRNA.

[0033] In certain embodiments, the composition comprises a total of 224 micrograms of mRNA.

[0034] In certain embodiments, the composition contains a total of 130 to 224 micrograms of mRNA.

[0035] In certain embodiments, the composition contains less than 100 micrograms of mRNA in total.

[0036] In certain embodiments, the composition contains less than 150 micrograms of mRNA in total.

[0037] In certain embodiments, the composition contains less than 200 micrograms of mRNA in total.

[0038] In certain embodiments, the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other.

[0039] In certain embodiments, the first mRNA, the second mRNA, the third mRNA, and / or the fourth mRNA are covalently linked to each other.

[0040] In certain embodiments, the first mRNA, the second mRNA, the third mRNA, and / or the fourth mRNA are incorporated into the LNP.

[0041] In certain embodiments, the LNP comprises at least one cationic lipid.

[0042] In certain embodiments, cationic lipids are biodegradable.

[0043] In certain embodiments, cationic lipids are not biodegradable.

[0044] In certain embodiments, cationic lipids are cleavable.

[0045] In certain embodiments, cationic lipids are not cleavable.

[0046] In certain embodiments, the cationic lipid is selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), and IM-001.

[0047] In certain embodiments, the cationic lipid is cKK-E10.

[0048] In certain embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

[0049] In certain embodiments, the cationic lipid is IM-001.

[0050] In certain embodiments, the LNP further comprises polyethylene glycol (PEG) conjugated lipids, cholesterol-based lipids, and helper lipids.

[0051] In certain embodiments, the LNP comprises cationic lipids in a molar ratio of 35% to 55%; polyethylene glycol (PEG) conjugated (PEGylated) lipids in a molar ratio of 0.25% to 2.75%; cholesterol-based lipids in a molar ratio of 20% to 45%; and helper lipids in a molar ratio of 5% to 35% (where all molar ratios are relative to the total lipid content of the LNP).

[0052] In certain embodiments, the LNP comprises a cationic lipid in a molar ratio of 40%, a PEGylated lipid in a molar ratio of 1.5%, a cholesterol-based lipid in a molar ratio of 28.5%, and a helper lipid in a molar ratio of 30% (where all molar ratios are relative to the total lipid content of the LNP).

[0053] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0054] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0055] In certain embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0056] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 40%; DMG-PEG2000 in a molar ratio of 1.5%; cholesterol in a molar ratio of 28.5%; and DOPE in a molar ratio of 30% (where all molar ratios are relative to the total lipid content of the LNP).

[0057] In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30% (where all molar ratios are relative to the total lipid content of the LNP).

[0058] In certain embodiments, the LNP comprises IM-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30% (where all molar ratios are relative to the total lipid content of the LNP).

[0059] In certain embodiments, the LNP has an average diameter of 30 nm to 200 nm.

[0060] In certain embodiments, the LNP has an average diameter of 80 nm to 150 nm.

[0061] In certain embodiments, the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A.

[0062] In certain embodiments, the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A.

[0063] In certain embodiments, the third mRNA encodes the HA antigen of the Victoria strain of influenza B.

[0064] In certain embodiments, the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B.

[0065] In certain embodiments, at least one mRNA contains a codon-optimized ORF.

[0066] In certain embodiments, at least one mRNA 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.

[0067] In certain embodiments, at least one mRNA molecule contains at least one chemical modification.

[0068] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one mRNA are chemically modified.

[0069] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one ORF are chemically modified.

[0070] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0071] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0072] In certain embodiments, the chemical modification is N1-methylpseuduridine.

[0073] In certain embodiments, the Disclosure provides a method for administering any of the above compositions to a subject in need thereof.

[0074] In certain embodiments, the Disclosure provides a method for inducing an immune response to influenza A or protecting a subject from influenza A infection, the method comprising administering one of the above compositions to the subject.

[0075] In certain embodiments, the Disclosure provides a method for inducing an immune response to influenza B or protecting a subject from influenza B infection, the method comprising administering one of the above compositions to the subject.

[0076] In certain embodiments, the serum concentration of neutralizing antibodies against influenza A is equivalent to that of a subject administered with a protein-type influenza vaccine, after administration of any of the above compositions.

[0077] In certain embodiments, subjects have serum concentrations of neutralizing antibodies against influenza B after administration of any of the above compositions, compared to subjects administered with a protein-based influenza B vaccine.

[0078] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A in the subject.

[0079] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza B in the subject.

[0080] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in the subject.

[0081] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in the subject.

[0082] In certain embodiments, the Disclosure provides compositions disclosed herein for use in methods of inducing an immune response to influenza A or protecting a subject from infection with influenza A.

[0083] In certain embodiments, the Disclosure provides compositions disclosed herein for use in methods of inducing an immune response to influenza B or protecting a subject from infection with influenza B.

[0084] In certain embodiments, the Disclosure provides the use of any of the compositions described herein in the manufacture of a pharmaceutical product for inducing an immune response to influenza A or for protecting a subject from infection with influenza A.

[0085] In certain embodiments, the Disclosure provides the use of any of the compositions described herein in the manufacture of a pharmaceutical product for inducing an immune response to influenza B or for protecting a subject from infection with influenza B.

[0086] In another embodiment, the present disclosure relates to a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encodes the HA antigen of a first influenza B virus, and the mRNA encoding the HA antigen of the influenza A virus is in a different ratio than the mRNA encoding the HA antigen of the influenza B virus. The composition provides lipid nanoparticles (LNPs) containing the following in w / w proportions: the first mRNA, the second mRNA, and the third mRNA are present in a 40% molar ratio: 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, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or IM-001; DMG-PEG2000 in a 1.5% molar ratio; cholesterol in a 28.5% molar ratio; and DOPE in a 30% molar ratio.

[0087] In one embodiment, the present disclosure relates to a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encodes the HA antigen of a first influenza B virus. The present invention provides a composition in which the mRNA encoding the HA antigen of influenza A virus is present in a different ratio (w / w) from the mRNA encoding the HA antigen of influenza B virus, and the first mRNA, second mRNA, and third mRNA are incorporated into lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%.

[0088] In one embodiment, the present disclosure is a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encodes the HA antigen of a first influenza B virus (wherein influenza A virus) The mRNA encoding the HA antigen of influenza B virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of influenza B virus, with the ratio of these mRNAs being 1:1:2 (w / w), and the composition is provided in which the first mRNA, second mRNA, and third mRNA are incorporated into lipid nanoparticles (LNPs) containing GL-HEPES-E3-E12-DS-4-E10 at a 40% molar ratio; DMG-PEG2000 at a 1.5% molar ratio; cholesterol at a 28.5% molar ratio; and DOPE at a 30% molar ratio.

[0089] In one embodiment, the Disclosure provides a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encodes the HA antigen of a first influenza B virus, wherein the mRNA encoding the HA antigen of the influenza A virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of the influenza B virus, and the first mRNA, the second mRNA, and the third mRNA are incorporated into lipid nanoparticles (LNPs) comprising IM-01 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0090] In one embodiment, the present disclosure is a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encodes the HA antigen of a first influenza B virus (type A). The present invention provides a composition in which the mRNA encoding the HA antigen of the influenza virus is present in a different ratio (w / w) from the mRNA encoding the HA antigen of the influenza B virus, with the ratio of these mRNAs being 1:1:2 (w / w), and the first mRNA, second mRNA, and third mRNA are incorporated into lipid nanoparticles (LNPs) containing IM-01 at a 40% molar ratio; DMG-PEG2000 at a 1.5% molar ratio; cholesterol at a 28.5% molar ratio; and DOPE at a 30% molar ratio.

[0091] In certain embodiments, the composition comprises a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different strains, and the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a 40% molar ratio of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPE It is incorporated into lipid nanoparticles (LNPs) containing S-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or IM-001; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE.

[0092] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 (w / w).

[0093] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:3 (w / w).

[0094] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:4 (w / w).

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

[0096] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:6 (w / w).

[0097] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:7 (w / w).

[0098] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:8 (w / w).

[0099] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:9 (w / w).

[0100] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:10 (w / w).

[0101] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w).

[0102] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:2:2 (w / w).

[0103] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:3:3 (w / w).

[0104] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:4:4 (w / w).

[0105] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:5:5 (w / w).

[0106] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:6:6 (w / w).

[0107] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:7:7 (w / w).

[0108] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:8:8 (w / w).

[0109] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:9:9 (w / w).

[0110] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:10:10 (w / w).

[0111] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 1:1:2:2 to approximately 1:1:10:10.

[0112] In certain embodiments, the ratio is expressed in micrograms (μg).

[0113] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 32 micrograms of the first mRNA, approximately 32 micrograms of the second mRNA, approximately 64 micrograms of the third mRNA, and approximately 64 micrograms of the fourth mRNA.

[0114] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 64 micrograms of the third mRNA, and approximately 64 micrograms of the fourth mRNA.

[0115] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 96 micrograms of the third mRNA, and approximately 96 micrograms of the fourth mRNA.

[0116] In certain embodiments, the composition contains less than 100 micrograms of mRNA in total.

[0117] In certain embodiments, the composition contains less than 150 micrograms of mRNA in total.

[0118] In certain embodiments, the composition contains less than 200 micrograms of mRNA in total.

[0119] In certain embodiments, the composition contains less than 250 micrograms of mRNA in total.

[0120] In certain embodiments, the composition contains a total of 100 to 250 micrograms of mRNA.

[0121] In certain embodiments, the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other.

[0122] In certain embodiments, the first mRNA, the second mRNA, the third mRNA, and / or the fourth mRNA are covalently linked to each other.

[0123] In certain embodiments, the LNP has an average diameter of 30 nm to 200 nm.

[0124] In certain embodiments, the LNP has an average diameter of 80 nm to 150 nm.

[0125] In certain embodiments, the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A.

[0126] In certain embodiments, the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A.

[0127] In certain embodiments, the third mRNA encodes the HA antigen of the Victoria strain of influenza B.

[0128] In certain embodiments, the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B.

[0129] In certain embodiments, at least one mRNA contains a codon-optimized ORF.

[0130] In certain embodiments, at least one mRNA 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.

[0131] In certain embodiments, at least one mRNA molecule contains at least one chemical modification.

[0132] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one mRNA are chemically modified.

[0133] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one ORF are chemically modified.

[0134] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0135] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0136] In certain embodiments, the chemical modification is N1-methylpseuduridine.

[0137] In certain embodiments, the Disclosure provides a method for administering any of the above compositions to a subject in need thereof.

[0138] In certain embodiments, the Disclosure provides a method for inducing an immune response to influenza A or protecting a subject from influenza A infection, the method comprising administering one of the above compositions to the subject.

[0139] In certain embodiments, the Disclosure provides a method for inducing an immune response to influenza B or protecting a subject from influenza B infection, the method comprising administering one of the above compositions to the subject.

[0140] In certain embodiments, the serum concentration of neutralizing antibodies against influenza A is equivalent to that of a subject administered with a protein-type influenza vaccine, after administration of any of the above compositions.

[0141] In certain embodiments, subjects have serum concentrations of neutralizing antibodies against influenza B after administration of any of the above compositions, compared to subjects administered with a protein-based influenza B vaccine.

[0142] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A in the subject.

[0143] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza B in the subject.

[0144] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in the subject.

[0145] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in the subject.

[0146] In certain embodiments, the Disclosure provides compositions disclosed herein for use in methods of inducing an immune response to influenza A or protecting a subject from infection with influenza A.

[0147] In certain embodiments, the Disclosure provides compositions disclosed herein for use in methods of inducing an immune response to influenza B or protecting a subject from infection with influenza B.

[0148] In certain embodiments, the Disclosure provides the use of any of the compositions described herein in the manufacture of a pharmaceutical product for inducing an immune response to influenza A or for protecting a subject from infection with influenza A.

[0149] In certain embodiments, the Disclosure provides the use of any of the compositions described herein in the manufacture of a pharmaceutical product for inducing an immune response to influenza B or for protecting a subject from infection with influenza B.

[0150] In another embodiment, the present disclosure is a method comprising administering to a human subject a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encodes the HA antigen of a first influenza B virus, and the mRNA encoding the HA antigen of the influenza A virus encodes the HA antigen of the influenza B virus. The present invention provides a method in which NA exists in different ratios (w / w), and the first mRNA, second mRNA, and third mRNA are incorporated into lipid nanoparticles (LNPs) containing 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, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or IM-001; DMG-PEG2000 at a 1.5% molar ratio; cholesterol at a 28.5% molar ratio; and DOPE at a 30% molar ratio.

[0151] In certain embodiments, the method comprises administering a composition comprising a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are different strains, and the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a 40% molar ratio of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, G It is incorporated into lipid nanoparticles (LNPs) containing L-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or IM-001; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE.

[0152] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 (w / w).

[0153] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:3 (w / w).

[0154] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:4 (w / w).

[0155] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:5 (w / w).

[0156] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:6 (w / w).

[0157] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:7 (w / w).

[0158] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:8 (w / w).

[0159] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:9 (w / w).

[0160] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:10 (w / w).

[0161] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w).

[0162] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:2:2 (w / w).

[0163] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:3:3 (w / w).

[0164] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:4:4 (w / w).

[0165] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:5:5 (w / w).

[0166] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:6:6 (w / w).

[0167] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:7:7 (w / w).

[0168] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:8:8 (w / w).

[0169] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:9:9 (w / w).

[0170] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:10:10 (w / w).

[0171] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 1:1:2:2 to approximately 1:1:10:10.

[0172] In certain embodiments, the ratio is expressed in micrograms (μg).

[0173] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 32 micrograms of the first mRNA, approximately 32 micrograms of the second mRNA, approximately 64 micrograms of the third mRNA, and approximately 64 micrograms of the fourth mRNA.

[0174] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 64 micrograms of the third mRNA, and approximately 64 micrograms of the fourth mRNA.

[0175] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 96 micrograms of the third mRNA, and approximately 96 micrograms of the fourth mRNA.

[0176] In certain embodiments, the composition contains less than 100 micrograms of mRNA in total.

[0177] In certain embodiments, the composition contains less than 150 micrograms of mRNA in total.

[0178] In certain embodiments, the composition contains less than 200 micrograms of mRNA in total.

[0179] In certain embodiments, the composition contains less than 250 micrograms of mRNA in total.

[0180] In certain embodiments, the composition contains a total of 100 to 250 micrograms of mRNA.

[0181] In certain embodiments, the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other.

[0182] In certain embodiments, the first mRNA, the second mRNA, the third mRNA, and / or the fourth mRNA are covalently linked to each other.

[0183] In certain embodiments, the LNP has an average diameter of 30 nm to 200 nm.

[0184] In certain embodiments, the LNP has an average diameter of 80 nm to 150 nm.

[0185] In certain embodiments, the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A.

[0186] In certain embodiments, the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A.

[0187] In certain embodiments, the third mRNA encodes the HA antigen of the Victoria strain of influenza B.

[0188] In certain embodiments, the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B.

[0189] In certain embodiments, at least one mRNA contains a codon-optimized ORF.

[0190] In certain embodiments, at least one mRNA 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.

[0191] In certain embodiments, at least one mRNA molecule contains at least one chemical modification.

[0192] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one mRNA are chemically modified.

[0193] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one ORF are chemically modified.

[0194] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0195] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0196] In certain embodiments, the chemical modification is N1-methylpseuduridine.

[0197] In certain embodiments, the composition is administered in an amount effective in inducing an immune response to influenza A or protecting the subject from influenza A infection.

[0198] In certain embodiments, the composition is administered in an amount effective in inducing an immune response to influenza B or protecting the subject from influenza B infection.

[0199] In certain embodiments, the subjects have serum concentrations of neutralizing antibodies against influenza A after administration of the composition, compared to subjects administered with a protein-based influenza A vaccine.

[0200] In certain embodiments, subjects have serum concentrations of neutralizing antibodies against influenza B after administration of the composition, compared to subjects administered with a protein-based influenza B vaccine.

[0201] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A in the subject.

[0202] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza B in the subject.

[0203] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in the subject.

[0204] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in the subject.

[0205] In another embodiment, the present disclosure relates to a composition comprising at least three messenger RNAs (mRNAs), wherein (i) a first mRNA encodes the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encodes the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) The third mRNA encodes the HA antigen of the first influenza B virus, and the first mRNA, the second mRNA, and the third mRNA are incorporated into lipid nanoparticles (LNPs) containing IM-001.

[0206] In certain embodiments, the composition comprises a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different strains, and the first mRNA, second mRNA, third mRNA, and fourth mRNA are incorporated into an LNP containing IM-001.

[0207] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 (w / w).

[0208] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:3 (w / w).

[0209] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:4 (w / w).

[0210] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:5 (w / w).

[0211] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:6 (w / w).

[0212] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:7 (w / w).

[0213] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:8 (w / w).

[0214] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:9 (w / w).

[0215] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:10 (w / w).

[0216] In certain embodiments, the composition comprises a first mRNA, a second mRNA, and a third mRNA, present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w).

[0217] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:2:2 (w / w).

[0218] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:3:3 (w / w).

[0219] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:4:4 (w / w).

[0220] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:5:5 (w / w).

[0221] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:6:6 (w / w).

[0222] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:7:7 (w / w).

[0223] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:8:8 (w / w).

[0224] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:9:9 (w / w).

[0225] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio of approximately 1:1:10:10 (w / w).

[0226] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 1:1:2:2 to approximately 1:1:10:10.

[0227] In certain embodiments, the ratio is expressed in micrograms (μg).

[0228] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 32 micrograms of the first mRNA, approximately 32 micrograms of the second mRNA, approximately 64 micrograms of the third mRNA, and approximately 64 micrograms of the fourth mRNA.

[0229] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 64 micrograms of the third mRNA, and approximately 64 micrograms of the fourth mRNA.

[0230] In certain embodiments, the composition comprises a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA, present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 96 micrograms of the third mRNA, and approximately 96 micrograms of the fourth mRNA.

[0231] In certain embodiments, the composition contains less than 100 micrograms of mRNA in total.

[0232] In certain embodiments, the composition contains less than 150 micrograms of mRNA in total.

[0233] In certain embodiments, the composition contains less than 200 micrograms of mRNA in total.

[0234] In certain embodiments, the composition contains less than 250 micrograms of mRNA in total.

[0235] In certain embodiments, the composition contains a total of 100 to 250 micrograms of mRNA.

[0236] In certain embodiments, the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other.

[0237] In certain embodiments, the first mRNA, the second mRNA, the third mRNA, and / or the fourth mRNA are covalently linked to each other.

[0238] In certain embodiments, the LNP further comprises polyethylene glycol (PEG) conjugated lipids, cholesterol-based lipids, and helper lipids.

[0239] In certain embodiments, the LNP comprises IM-001 in a molar ratio of 35% to 55%; polyethylene glycol (PEG) conjugate (PEGylated) lipids in a molar ratio of 0.25% to 2.75%; cholesterol-based lipids in a molar ratio of 20% to 45%; and helper lipids in a molar ratio of 5% to 35% (where all molar ratios are relative to the total lipid content of the LNP).

[0240] In certain embodiments, the LNP comprises 40% molar ratio of IM-001; 1.5% molar ratio of PEGylated lipids; 28.5% molar ratio of cholesterol-based lipids; and 30% molar ratio of helper lipids (where all molar ratios are relative to the total lipid content of the LNP).

[0241] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0242] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0243] In certain embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0244] In certain embodiments, the LNP has an average diameter of 30 nm to 200 nm.

[0245] In certain embodiments, the LNP has an average diameter of 80 nm to 150 nm.

[0246] In certain embodiments, the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A.

[0247] In certain embodiments, the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A.

[0248] In certain embodiments, the third mRNA encodes the HA antigen of the Victoria strain of influenza B.

[0249] In certain embodiments, the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B.

[0250] In certain embodiments, at least one mRNA contains a codon-optimized ORF.

[0251] In certain embodiments, at least one mRNA 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.

[0252] In certain embodiments, at least one mRNA molecule contains at least one chemical modification.

[0253] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one mRNA are chemically modified.

[0254] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in at least one ORF are chemically modified.

[0255] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0256] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0257] In certain embodiments, the chemical modification is N1-methylpseudridine.

[0258] In certain embodiments, the Disclosure provides a method for administering any of the above compositions to a subject in need thereof.

[0259] In certain embodiments, the Disclosure provides a method for inducing an immune response to influenza A or protecting a subject from influenza A infection, the method comprising administering one of the above compositions to the subject.

[0260] In certain embodiments, the Disclosure provides a method for inducing an immune response to influenza B or protecting a subject from influenza B infection, the method comprising administering one of the above compositions to the subject.

[0261] In certain embodiments, the serum concentration of neutralizing antibodies against influenza A is equivalent to that of a subject administered with a protein-type influenza vaccine, after administration of any of the above compositions.

[0262] In certain embodiments, subjects have serum concentrations of neutralizing antibodies against influenza B after administration of any of the above compositions, compared to subjects administered with a protein-based influenza B vaccine.

[0263] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A in the subject.

[0264] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza B in the subject.

[0265] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in the subject.

[0266] In certain embodiments, the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in the subject.

[0267] In certain embodiments, the Disclosure provides compositions disclosed herein for use in methods of inducing an immune response to influenza A or protecting a subject from infection with influenza A.

[0268] In certain embodiments, the Disclosure provides compositions disclosed herein for use in methods of inducing an immune response to influenza B or protecting a subject from infection with influenza B.

[0269] In certain embodiments, the Disclosure provides the use of any of the compositions described herein in the manufacture of a pharmaceutical product for inducing an immune response to influenza A or for protecting a subject from infection with influenza A.

[0270] In certain embodiments, the Disclosure provides the use of any of the compositions described herein in the manufacture of a pharmaceutical product for inducing an immune response to influenza B or for protecting a subject from infection with influenza B.

[0271] The above and other features and advantages of this disclosure will be better understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0272] [Figure 1]Figure 1 shows the interim immunogenicity data from a Phase I / II trial (clinical trial NCT05624606) investigating the influenza vaccine MRT5410, a quadrivalent modified RNA influenza vaccine encapsulated in lipid nanoparticles (LNPs) containing the cationic lipid GL-HEPES-E3-E12-DS-4-E10, encoding the hemagglutinin (HA) sequences of two influenza A strains (i.e., A / H1N1 and A / H3N2) and two influenza B strains (i.e., B / Yamagata lineage and B / Victoria lineage) in a 1:1:1:1 ratio. The vaccine was administered as a single intramuscular injection to adults aged 18–64 years and compared to the following active controls: (1) Fluzone Quadrivalent® (quadrivalent inactivated standard-dose influenza vaccine); and (2) Flublok Quadrivalent® (quadrivalent recombinant influenza vaccine). Hemagglutinin inhibition (HAI) geometric mean titer ratios (GMTRs) and serum conversion percentages were measured for influenza strains of the A / H1N1, A / H3N2, B / Yamagata lineage, and B / Victoria lineage in each vaccine. [Figure 2] Figure 2 shows intermediate immunogenicity data from a Phase I / II trial (clinical trial NCT05553301) investigating influenza vaccine MRT5410, a quadrivalent modified RNA influenza vaccine encapsulated in an LNP formulation containing the cationic lipid ckk-E10, encoding the HA sequences of two influenza A strains (i.e., A / H1N1 and A / H3N2) and two influenza B strains (i.e., B / Yamagata lineage and B / Victoria lineage) in a 1:1:1:1 ratio. The vaccine was administered as a single intramuscular injection at two dose levels (i.e., dose level 1 and dose level 2) to adults aged 18–64 years and compared to the following active controls: (1) Fluzone Quadrivalent® (quadrivalent inactivated standard-dose influenza vaccine); and (2) Flublok Quadrivalent® (quadrivalent recombinant influenza vaccine). Hemagglutinin inhibition (HAI) geometric mean titer ratios (GMTRs) and serum conversion percentages were measured for influenza strains of the A / H1N1, A / H3N2, B / Yamagata lineage, and B / Victoria lineage in each vaccine. [Modes for carrying out the invention]

[0273] This disclosure relates, in particular, to novel RNA (e.g., mRNA) compositions encoding combinations of influenza A and influenza B hemagglutinin (HA) antigens, wherein the mRNA encoding the HA antigen of influenza A virus is present in a different ratio (w / w) than the mRNA encoding the influenza B virus, and to methods for inducing an immune response by administering such compositions. In particular, this disclosure relates to mRNA encoding these antigens incorporated into lipid nanoparticles (LNPs).

[0274] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of the present invention. In case of any inconsistency, this specification shall prevail, including definitions. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. Enzyme reactions and purification techniques shall be carried out according to the manufacturer's specifications, as commonly performed in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include plurals, and plural terms shall include singulars. Throughout this specification and its embodiments, the terms “having” and “including,” or variations such as “having,” “having,” “including,” or “containing,” are understood to mean including the integer or group of integers described, but not to mean excluding any other integer or group of integers. All publications and other references referenced herein are incorporated in their entirety by reference. This specification references several documents, but such references do not constitute an endorsement that any of those documents form part of the common technical knowledge in the art.

[0275] It should be noted that the term “a (a)” or “an (an)” entity refers to one or more of those entities; for example, “a nucleotide sequence” is understood to represent one or more nucleotide sequences. Therefore, the terms “a (a)” (or “an (an)”), “one or more,” and “at least one” can be used interchangeably herein.

[0276] Furthermore, as used herein, “and / or” should be understood to specifically disclose each of the two designated features or components, with or without the other. Thus, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to encompass each of the following embodiments: 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).

[0277] Whenever an aspect is described herein with the word "comprising," it should be understood that similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0278] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to whom this disclosure relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide a general dictionary of many of the terms used herein.

[0279] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges include the numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right with an amino-to-carboxyl orientation. The headings provided herein are not limitations on the various aspects of this disclosure. Thus, terms defined immediately thereafter are defined in more detail by referring to this specification as a whole.

[0280] The terms “approximately” or “about” are used herein to mean roughly, broadly, approximately, or within a range. When used in conjunction with a numerical range, the term “about” modifies the range by extending the boundary above or below the indicated number. Generally, the term “about” can modify a number that is above or below a given value by, for example, a difference of 10 percent. In some embodiments, the term indicates a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the indicated number. In some embodiments, “about” indicates a deviation of ±10% from the indicated number. In some embodiments, “about” indicates a deviation of ±5% from the indicated number. In some embodiments, "approximately" indicates a deviation of ±4% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±2% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.01% from the given value.

[0281] As used herein, the terms “messenger RNA” or “mRNA” refer to a polynucleotide encoding 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 also substituted for the open reading frame (ORF). The non-coding region of mRNA includes the 5' cap, 5' untranslated region (UTR), 3'UTR, and poly(A) tail. mRNA can be purified from natural sources, produced using recombinant expression systems (e.g., in vitro transcription), and optionally purified or chemically synthesized.

[0282] As used herein, the term “polypeptide” refers to any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers, including natural and non-natural amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids, such as artificial chemical mimics of the corresponding natural amino acids. “Residue” refers to an amino acid or amino acid mimic incorporated into a polypeptide by an amide bond or amide bond mimic. Polypeptides have an amino terminus (N-terminus) and a carboxyl terminus (C-terminus). “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

[0283] 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 may include any cells in the body involved in host defense reactions, such as epithelial cells that secrete interferons or cytokines. An immune response may include, but is not limited to, innate and / or adaptive immune responses.

[0284] As used herein, “protective immune response” refers to an immune response that protects an object from infection (e.g., preventing infection or the development of an infection-related disease). Methods for measuring immune responses include, for example, measuring the proliferation and / or activity of lymphocytes (such as B cells or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc.

[0285] As used herein, “antibody response” refers to an immune response in which antibodies are produced.

[0286] As used herein, “antigen” refers to a drug that, when exposed to or administered to an organism, elicits an immune response, and / or a drug that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Alternatively, or further, in some embodiments, an antigen elicits a cellular response in an organism (e.g., involving a T cell whose receptor specifically interacts 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 species. In some embodiments, the 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 species. In some embodiments, the antigen may or may not bind to antibodies and / or T cell receptors and induce specific physiological responses in the organism. In some embodiments, for example, the antigen may bind to antibodies and / or T cell receptors in vitro, regardless of whether such interactions occur in vivo. In some embodiments, the antigen reacts with products of specific humoral or cellular immunity. Antigens include hemagglutinin (HA) antigens of both influenza A virus, influenza B virus, or other influenza viruses described herein.

[0287] As used herein, “adjuvant” refers to a substance or vehicle that enhances the immune response to an antigen. Examples of adjuvants include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which an antigen has been adsorbed; and water-in-oil or oil-in-water emulsions in which an antigen solution is emulsified in mineral oil or water (e.g., Freund’s incomplete adjuvant). Sometimes, dead mycobacteria are included to further enhance antigenicity (e.g., Freund’s complete adjuvant). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants may also include biological molecules such as Toll-like receptor (TLR) agonists and co-stimulatory molecules.

[0288] As used herein, “Subject” refers to any member of the animal kingdom. In some embodiments, “Subject” refers to a human being. In some embodiments, “Subject” refers to a non-human animal. In some embodiments, the subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, the subject may be a transgenic animal, a genetically modified animal, and / or a clone. In certain embodiments, the subject is an adult, adolescent, or infant. In some embodiments, the terms “individual” or “patient” are used and are intended to be interchangeable with “subject.” In certain exemplary embodiments, the subjects are premature infants (e.g., gestational age less than 37 weeks), newborns (e.g., 0 to 27 days old), infants or toddlers (e.g., 28 to 23 months old), children (e.g., 2 to 11 years old), adolescents (e.g., 12 to 17 years old), adults (e.g., 18 to 50 years old or 18 to 64 years old), or elderly (e.g., 65 years and older). In exemplary embodiments, the subjects are 18 to 50 years old. In other exemplary embodiments, the subjects are elderly (e.g., adults 60 years and older).

[0289] As used herein, the terms “vaccination” or “to vaccinate” refer to the administration of a composition intended to produce an immune response to a disease-causing agent, for example. Vaccination can be performed before, during, and / or after exposure to a disease-causing agent, and / or before, during, and / or after the onset of one or more symptoms, and in some embodiments, before, during, and / or immediately after exposure to a disease-causing agent. In some embodiments, vaccination involves multiple administrations of the vaccine composition at appropriate time intervals.

[0290] This disclosure describes nucleic acid sequences (e.g., DNA and RNA sequences) and amino acid sequences that have a certain degree of identity with respect to a given nucleic acid sequence or amino acid sequence (reference sequence).

[0291] II.RNA The vaccines of this disclosure may comprise at least three ribonucleic acid (RNA) molecules, each containing an ORF encoding the influenza hemagglutinin (HA) antigen of influenza A and / or influenza B virus. In certain embodiments, the RNA is a messenger RNA (mRNA) comprising an ORF encoding the HA antigen of a first influenza A virus, an HA antigen of a second influenza A virus (wherein the first and second influenza A viruses are distinct subtypes), and an HA antigen of an influenza B virus. In further embodiments, the RNA is an mRNA comprising an ORF encoding the HA antigen of a first influenza A virus, an HA antigen of a second influenza A virus (wherein the first and second influenza A viruses are distinct subtypes), an HA antigen of a first influenza B virus, and an HA antigen of a second influenza B virus (wherein the first and second influenza B viruses are distinct lineages). In certain embodiments, the RNA (e.g., mRNA) further comprises at least one 5'UTR, 3'UTR, poly(A) tail, and / or 5' cap.

[0292] A.5' Cap The 5' cap of mRNA can provide resistance to nucleases found in most eukaryotic cells and can enhance translational efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also called "m7G" or "cap 0") contains guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0293] The 5’ cap is typically added as follows: First, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyl transferase, generating a 5’5’5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyl transferase. 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. Further cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0294] 5'-capping of polynucleotides can be accomplished simultaneously during in vitro transcription reactions using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 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). Vaccinia virus capping enzyme can be used to complete 5'-capping of the modified RNA post-transcriptionally to generate a cap 0 structure: m7G(5')ppp(5')G. Both vaccinia virus capping enzyme and 2'-O-methyl-transferase can be used to generate a cap 1 structure to generate 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 third 5'-nucleotide from the end using 2'-O-methyl-transferase. A cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the fourth 5'-nucleotide from the end using 2'-O-methyl-transferase.

[0295] In certain embodiments, the mRNA of the present disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; and m7G(5')ppp(5')(2'OMeG)pG.

[0296] In certain embodiments, the mRNA of the present disclosure comprises the following 5' cap: [ka] .

[0297] B. Untranslated Region (UTR) In some embodiments, the mRNA of this disclosure includes a 5' and / or 3' untranslated region (UTR). In mRNA, the 5' UTR begins at the transcription start site and continues to the start codon, but does not contain the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0298] In some embodiments, the mRNA disclosed herein may include a 5'UTR containing one or more elements that affect mRNA stability or translation. In some embodiments, the 5'UTR may be about 10 to 5,000 nucleotides long. In some embodiments, the 5'UTR may be about 50 to 500 nucleotides long. In some embodiments, the 5'UTR may be at least about 10 nucleotides long, about 20 nucleotides long, about 30 nucleotides long, about 40 nucleotides long, about 50 nucleotides long, about 100 nucleotides long, about 150 nucleotides long, about 200 nucleotides long, about 250 nucleotides long, about 300 nucleotides long, about 350 nucleotides long, about 400 nucleotides long, about 450 nucleotides long, about 500 nucleotides long, about 550 nucleotides long, about 600 nucleotides long, about 6 The lengths are approximately 50 nucleotides, 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.

[0299] In some embodiments, the mRNA disclosed herein may include a 3'UTR comprising one or more polyadenylation signals, protein binding sites affecting the stability of mRNA location in cells, or one or more binding sites to miRNAs. In some embodiments, the 3'UTR may be 50 to 5,000 nucleotides or longer. In some embodiments, the 3'UTR may be 50 to 1,000 nucleotides or longer. In some embodiments, the 3'UTR is at least about 50 nucleotides long, about 100 nucleotides long, about 150 nucleotides long, about 200 nucleotides long, about 250 nucleotides long, about 300 nucleotides long, about 350 nucleotides long, about 400 nucleotides long, about 450 nucleotides long, about 500 nucleotides long, about 550 nucleotides long, about 600 nucleotides long, about 650 nucleotides long, about 700 nucleotides long, about 750 nucleotides long, about 800 nucleotides long, about 850 nucleotides long, about 900 nucleotides long, about 950 nucleotides long, about 1,000 nucleotides long, about 1,500 nucleotides long, about 2,000 nucleotides long, about 2,500 nucleotides long, about 3,000 nucleotides long, about 3,500 nucleotides long, about 4,000 nucleotides long, about 4,500 nucleotides long, or about 5,000 nucleotides long.

[0300] In some embodiments, the mRNA disclosed herein may include 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).

[0301] In certain embodiments, the 5' and / or 3'UTR sequences may be derived from stable mRNA to enhance mRNA stability (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzymes). For example, the 5'UTR sequence may contain a sub-sequence or fragment thereof of the pre-initial 1 (IE1) gene to improve nuclease resistance and / or improve mRNA half-life. It is also conceivable to include a sequence or fragment thereof encoding human growth hormone (hGH) at the 3' end or untranslated region of the mRNA. Generally, these modifications improve mRNA stability and / or pharmacokinetic properties (e.g., half-life) compared to the unmodified counterpart, including modifications made, for example, to improve resistance of such mRNA to in vivonuclease digestion.

[0302] Examples of 5'UTRs include sequences derived from the pre-initial 1 (IE1) CMV gene (U.S. Patent Application Publication No. 2014 / 0206753 and U.S. Patent Application Publication No. 2015 / 0157565, which are incorporated herein by reference, respectively) or the sequence GGGAUCCUACC (Sequence ID 1) (U.S. Patent Application Publication No. 2016 / 0151409, which is incorporated herein by reference).

[0303] In various embodiments, the 5'UTR may be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks the 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publications 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, which are incorporated herein by reference, respectively).

[0304] In certain embodiments, the 5’UTR is derived from the ribosomal protein large 32 (L32) gene (U.S. Patent Application Publication No. 2017 / 0029847).

[0305] In certain embodiments, the 5’UTR is derived from the 5’UTR of the hydroxysteroid (17 - b) dehydrogenase 4 gene (HSD17B4) (U.S. Patent Application Publication No. 2016 / 0166710).

[0306] In certain embodiments, the 5’UTR is derived from the 5’UTR of the ATP5A1 gene (U.S. Patent Application Publication No. 2016 / 0166710).

[0307] In some embodiments, an internal ribosome entry site (IRES) is used instead of the 5’UTR.

[0308] In some embodiments, the 5’UTR is

Chemical formula

[0309] In some embodiments, the 3’UTR is

Chemical formula

[0310] The 5’UTR and 3’UTR are described in more detail in International Publication No. 2012 / 075040 pamphlet, which is incorporated herein by reference.

[0311] C. Polyadenylation tail As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to the sequence of adenosine nucleotides at the 3' end of an mRNA molecule. Poly(A) tails can confer stability to mRNA and protect it from exonuclease degradation. Poly(A) tails can enhance translation. In some embodiments, poly(A) tails are substantially homopolymers. For example, a poly(A) tail of 100 adenosine nucleotides may have a length of substantially 100 nucleotides. In certain embodiments, a poly(A) tail may be interrupted by at least one nucleotide different from adenosine nucleotides (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length greater than 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide different from adenosine nucleotides or a stretch of nucleotides). In certain embodiments, the poly(A) tail is a sequence: [ka] Includes an array of.

[0312] As used herein, “poly(A)tail” typically refers to RNA. However, in relation to this disclosure, the term also refers to the corresponding sequence in a DNA molecule (e.g., “poly(T) sequence”).

[0313] The poly(A) tail may contain approximately 10 to 500 adenosine nucleotides, approximately 10 to 200 adenosine nucleotides, approximately 40 to 200 adenosine nucleotides, or approximately 40 to 150 adenosine nucleotides. The length of the poly(A) tail may be at least approximately 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0314] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from the DNA template during in vitro transcription of the RNA. In certain embodiments, the poly(A) tail is obtained in vitro by common chemical synthesis methods without transcription from the DNA template. In various embodiments, the poly(A) tail is produced 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) polymerase, for example, using the methods and means described in International Publication No. 2016 / 174271.

[0315] Nucleic acids may contain poly(A) tails obtained by enzymatic polyadenylation, and most nucleic acid molecules contain approximately 100 (+ / -20) to 500 (+ / -50) or 250 (+ / -20) adenosine nucleotides.

[0316] In some embodiments, the nucleic acid may include a poly(A) tail derived from template DNA, as described, for example, in International Publication No. 2016 / 091391, and may further include at least one additional poly(A) tail generated by enzymatic polyadenylation. It is produced using the method described in Publication No. 2016 / 091391.

[0317] In certain embodiments, the nucleic acid includes at least one polyadenylation signal.

[0318] In various embodiments, the nucleic acid may contain at least one poly(C) sequence.

[0319] As used herein, the term “poly(C) sequence” is intended to be a sequence of cytosine nucleotides up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0320] D. Chemical modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may include at least one chemical modification. In some embodiments, the mRNA disclosed herein may include one or more modifications that typically enhance RNA stability. Exemplary modifications include skeletal 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), for example, but not limited to purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA is a modified nucleotide analog or derivative of purines and pyrimidines, for example, 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-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxymethylaminomethyl-2-thiouracil) It can be synthesized from roxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-pseuduracil, keosin, β-D-mannosylkeosin, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine, etc.

[0321] In some embodiments, the disclosed mRNA may include at least one chemical modification, for example, but are not limited to pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0322] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0323] In some embodiments, the chemical modification includes N1-methylpseuduridine.

[0324] 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.

[0325] 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.

[0326] Preparations of such analogues are 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.

[0327] E.mRNA synthesis The mRNA disclosed herein may be synthesized according to any of the following methods. For example, the mRNA according to this disclosure may be synthesized via in vitro transcription (IVT). Several methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin.Immunol. 25(2):152-159; and Brunelle et al. (2013) Methods Enzymol. 530:101-14. Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which may include DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or an RNase inhibitor. The exact conditions may vary depending on the specific application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. In some embodiments, mRNA provided from an in vitro transcription reaction may be preferred, but other mRNA sources, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals, can be used in accordance with this disclosure.

[0328] III. Combination influenza mRNA vaccine compositions Influenza is a negative-sense single-stranded RNA virus belonging to the Orthomyxoviridae family. Every year, influenza viruses affect millions of people worldwide, causing significant mortality and morbidity. Hemagglutinin (HA) is one of the major glycoproteins on the surface of the influenza virus and is essential for influenza infectivity. HA plays a major role in the attachment of the influenza virus to host cells and the fusion of the viral membrane with the host membrane.

[0329] HA consists of two subunits: HA1 and HA2. For HA to be active as a fusion protein, it must be cleaved by a cellular protease. Cleavage of HA0 into HA1 and HA2 activates viral infectivity and is crucial for influenza virus pathogenicity in humans.

[0330] RNA (e.g., mRNA) compositions targeting three or more influenza HA antigens are provided herein.

[0331] In some embodiments, the composition comprises mRNA encoding a polypeptide derived from an influenza virus protein selected from hemagglutinin (e.g., hemagglutinin 1 (HA1) and hemagglutinin 2 (HA2), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2)). In further embodiments, the composition comprises mRNA encoding a polypeptide derived from the HA protein, the NA protein, and both the HA protein and the NA protein. In other embodiments, the composition comprises mRNA encoding an antigenic polypeptide derived from a different influenza strain.

[0332] In some embodiments, the composition comprises mRNA encoding antigens of influenza A, B, and / or C type viruses. In certain embodiments, the composition comprises mRNA encoding HA and / or NA antigens of influenza A and B type viruses. In some embodiments, the HA antigen of influenza A virus is selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. In other embodiments, the NA antigen of influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. In certain embodiments, the HA and NA antigens of influenza B virus are derived from the influenza B / Yamagata lineage. In some embodiments, the HA and NA antigens of influenza B virus are derived from the influenza B / Victoria lineage. In some embodiments, the HA and / or NA antigens are derived from influenza virus strains recommended in the World Health Organization's (WHO) annual recommendations for influenza vaccine formulations.

[0333] In certain embodiments, at least one of one or more influenza virus proteins comprises an influenza virus HA protein having a molecular sequence identified or designed from a machine learning model, and in certain embodiments, at least one of one or more ribonucleic acid molecules encodes one or more influenza virus proteins having a molecular sequence identified or designed from a machine learning model.

[0334] In one embodiment, the composition comprises one mRNA encoding the H1 HA antigen, one mRNA encoding the H3 HA antigen, one mRNA encoding the HA antigen derived from the influenza B / Yamagata lineage, and one mRNA encoding the HA antigen derived from the influenza B / Victoria lineage.

[0335] In one embodiment, the composition comprises one mRNA encoding an H1 HA antigen, one mRNA encoding an H3 HA antigen, and one HA antigen derived from the influenza B / Victoria lineage.

[0336] In another embodiment, the composition further comprises one or more mRNA molecules encoding machine learning influenza virus HA having a molecular sequence identified or designed from a machine learning model, the one or more machine learning influenza virus HAs may be selected from H1 HA, H3 HA, HA from the B / Victoria lineage, HA from the B / Yamagata lineage, or a combination thereof.

[0337] When selecting one or more machine learning influenza virus HAs, any machine learning algorithm may be used. For example, as assumed herein, any of the machine learning algorithms and methods disclosed in PCT application International Publication No. 2021 / 080990A1 (Title of Invention: Systems and Methods for Designing Vaccines) and International Publication No. 2021 / 080999A1 (Title of Invention: Systems and Methods for Predicting Biological Responses) (both of which are incorporated herein by reference in their entirety).

[0338] mRNA may be unmodified (i.e., containing only native ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or chemically modified (e.g., containing nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudridine), 2'-fluororibonucleotide, and 2'-methoxyribonucleotide, and / or phosphorothioate bonds). mRNA molecules may include a 5' cap and a polyA tail.

[0339] In one embodiment, the composition comprises at least three messenger RNAs (mRNAs), the at least three mRNAs comprising an open reading frame (ORF) encoding an HA antigen selected from the group consisting of (i) a first mRNA encoding the hemagglutinin (HA) antigen of a first influenza A virus; (ii) a second mRNA encoding the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encoding the HA antigen of a first influenza B virus (where the mRNA encoding the HA antigen of the influenza A virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of the influenza B virus).

[0340] In some embodiments, the composition comprises at least three mRNAs: (i) a first mRNA encoding the HA antigen of a first influenza A virus; (ii) a second mRNA encoding the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encoding the HA antigen of a first influenza B virus, with the mRNA encoding the HA antigen of the influenza A virus present in a different ratio (w / w) from the mRNA encoding the HA antigen of the influenza B virus. The first mRNA, second mRNA, and third mRNA are present in lipid nanoparticles (LNPs) containing 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, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or IM-001; DMG-PEG2000 at a 1.5% molar ratio; cholesterol at a 28.5% molar ratio; and DOPE at a 30% molar ratio.

[0341] In some embodiments, the composition comprises at least three mRNAs: (i) a first mRNA encoding the HA antigen of a first influenza A virus; (ii) a second mRNA encoding the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) a third mRNA encoding the HA antigen of a first influenza B virus, wherein the first mRNA, the second mRNA, and the third mRNA are incorporated into an LNP containing IM-001.

[0342] Any of the embodiments described herein may contain a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different strains.

[0343] Any of the embodiments described herein may comprise a first mRNA, a second mRNA, a third mRNA, and / or a fourth mRNA that are not covalently bonded to each other.

[0344] Any of the embodiments described herein may comprise a first mRNA, a second mRNA, a third mRNA, and / or a fourth mRNA that are covalently bound to each other.

[0345] Any of the embodiments described herein may contain a first mRNA encoding the HA antigen of the H1N1 subtype of influenza A.

[0346] Any of the embodiments described herein may contain a second mRNA encoding the HA antigen of the influenza A H3N2 subtype.

[0347] Any one of the embodiments described herein may comprise a third mRNA encoding the HA antigen of the Victoria strain of influenza B.

[0348] Any one of the embodiments described herein may contain a fourth mRNA encoding the HA antigen of the Yamagata strain of influenza B.

[0349] Any of the embodiments described herein may comprise at least one mRNA containing a codon-optimized ORF.

[0350] A composition of any embodiment described herein may comprise at least one mRNA comprising at least one 5'UTR, at least one 3'UTR, and at least one poly(A) sequence.

[0351] Any of the embodiments described herein may comprise at least one mRNA having at least one chemical modification.

[0352] The compositions of any embodiment described herein may contain 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 chemically modified uracil nucleotides in at least one mRNA.

[0353] The compositions of any embodiment described herein may contain 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 chemically modified uracil nucleotides in at least one ORF.

[0354] A composition of any embodiment described herein may include a chemical modification selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0355] Any of the compositions of the embodiments described herein may include chemical modifications selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0356] Any of the embodiments described herein may contain chemically modified N1-methylpseudridine.

[0357] Any of the embodiments described herein may comprise a first mRNA, a second mRNA, a third mRNA, and / or a fourth mRNA incorporated into the LNP.

[0358] Any of the embodiments described herein may include an LNP containing at least one cationic lipid.

[0359] Any of the embodiments described herein may include LNPs in which the cationic lipid is biodegradable.

[0360] The compositions of any embodiment described herein may include LNPs in which the cationic lipid is not biodegradable.

[0361] Any of the embodiments described herein may include LNPs on which cationic lipids can be cleaved.

[0362] Any of the embodiments described herein may include LNPs in which the cationic lipids are not cleavable.

[0363] A composition of any embodiment described herein may contain an LNP 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, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), and IM-001, in which the cationic lipid is selected.

[0364] Any of the embodiments described herein may include an LNP in which the cationic lipid is cKK-E10.

[0365] Any of the embodiments described herein may contain an LNP in which the cationic lipid is GL-HEPES-E3-E12-DS-4-E10.

[0366] Any of the embodiments described herein may include an LNP in which the cationic lipid is IM-001.

[0367] Any of the embodiments described herein may further comprise LNPs comprising ethylene glycol (PEG) conjugate (PEGylated) lipids, cholesterol-based lipids, and helper lipids.

[0368] Any of the embodiments described herein may contain LNP, which consists of a cationic lipid in a molar ratio of 35% to 55%; polyethylene glycol (PEG) conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75%; cholesterol-based lipids in a molar ratio of 20% to 45%; and helper lipids in a molar ratio of 5% to 35% (wherein all molar ratios are relative to the total lipid content of the LNP).

[0369] Any of the embodiments described herein may comprise an LNP consisting of a cationic lipid in a molar ratio of 40%, a PEGylated lipid in a molar ratio of 1.5%, a cholesterol-based lipid in a molar ratio of 28.5%, and a helper lipid in a molar ratio of 30% (wherein all molar ratios are relative to the total lipid content of the LNP).

[0370] A composition of any embodiment described herein may contain an LNP in which the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0371] Any of the embodiments described herein may contain an LNP in which the cholesterol lipid is cholesterol.

[0372] A composition of any embodiment described herein may include an LNP in which the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0373] Any of the embodiments described herein may comprise LNP, which consists of GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 40%; DMG-PEG2000 in a molar ratio of 1.5%; cholesterol in a molar ratio of 28.5%; and DOPE in a molar ratio of 30% (wherein all molar ratios are relative to the total lipid content of the LNP).

[0374] Any of the embodiments described herein may comprise LNP, which consists of cKK-E10 in a molar ratio of 40%; DMG-PEG2000 in a molar ratio of 1.5%; cholesterol in a molar ratio of 28.5%; and DOPE in a molar ratio of 30% (wherein all molar ratios are relative to the total lipid content of the LNP).

[0375] Any of the embodiments described herein may comprise LNP, which consists of IM-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30% (wherein all molar ratios are relative to the total lipid content of the LNP).

[0376] Any of the compositions of the embodiments described herein may include LNPs having an average diameter of 30 nm to 200 nm.

[0377] Any of the embodiments described herein may include LNPs having an average diameter of 80 nm to 150 nm.

[0378] Section IV of this specification further describes LNPs that can be formulated together with the combination influenza mRNA vaccine compositions discussed herein and incorporated herein.

[0379] A. Ratio of influenza HA mRNA As discussed in the examples, current mRNA-based polyvalent influenza vaccines under investigation encode equal amounts (w / w) of hemagglutinin (HA) antigens of both influenza A and influenza B viruses. While these vaccines are immunogenic, ongoing trials have shown that the immunogenicity of strain B is lower than that of strain A, and that the human immune response to influenza B virus is not optimal with these vaccines. One exemplary strategy to improve the immune response to strain B is to increase the amount / ratio of mRNA encoding the HA sequence of influenza B virus compared to mRNA encoding the HA sequence of influenza A virus.

[0380] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of approximately 1:1:2.

[0381] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of approximately 1:1:3.

[0382] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio (w / w) of approximately 1:1:4.

[0383] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of approximately 1:1:5.

[0384] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of approximately 1:1:6.

[0385] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of approximately 1:1:7.

[0386] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio (w / w) of approximately 1:1:8.

[0387] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio (w / w) of approximately 1:1:9.

[0388] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio (w / w) of approximately 1:1:10.

[0389] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in a 1:1:2 mRNA ratio (w / w).

[0390] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:3.

[0391] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:4.

[0392] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:5 (w / w).

[0393] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:6.

[0394] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:7.

[0395] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:8.

[0396] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:9.

[0397] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio (w / w) of 1:1:10.

[0398] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:2 (w / w).

[0399] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:3 (w / w).

[0400] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:4 (w / w).

[0401] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:5 (w / w).

[0402] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:6 (w / w).

[0403] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:7 (w / w).

[0404] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:8 (w / w).

[0405] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:9 (w / w).

[0406] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of approximately 1:1:10 (w / w).

[0407] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:2 (w / w).

[0408] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:3 (w / w).

[0409] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:4 (w / w).

[0410] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:5 (w / w).

[0411] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:6 (w / w).

[0412] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:7 (w / w).

[0413] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:8 (w / w).

[0414] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:9 (w / w).

[0415] Any of the embodiments described herein may contain a first mRNA, a second mRNA, and a third mRNA in a ratio of 1:1:10 (w / w).

[0416] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of about 1:1:2 to about 1:1:10 (w / w).

[0417] Any of the embodiments described herein may comprise a first mRNA, a second mRNA, and a third mRNA in an mRNA ratio expressed in micrograms (μg).

[0418] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:2:2.

[0419] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:3:3.

[0420] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:4:4.

[0421] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:5:5.

[0422] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:6:6.

[0423] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:7:7.

[0424] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:8:8.

[0425] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:9:9.

[0426] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of approximately 1:1:10:10.

[0427] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:2:2.

[0428] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:3:3.

[0429] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:4:4.

[0430] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:5:5.

[0431] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:6:6.

[0432] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:7:7.

[0433] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:8:8.

[0434] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:9:9.

[0435] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio (w / w) of 1:1:10:10.

[0436] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:2:2 (w / w).

[0437] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:3:3 (w / w).

[0438] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:4:4 (w / w).

[0439] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:5:5 (w / w).

[0440] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:6:6 (w / w).

[0441] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:7:7 (w / w).

[0442] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:8:8 (w / w).

[0443] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:9:9 (w / w).

[0444] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:10:10 (w / w).

[0445] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:2:2 (w / w).

[0446] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:3:3 (w / w).

[0447] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:4:4 (w / w).

[0448] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:5:5 (w / w).

[0449] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:6:6 (w / w).

[0450] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:7:7 (w / w).

[0451] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:8:8 (w / w).

[0452] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:9:9 (w / w).

[0453] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:10:10 (w / w).

[0454] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of about 1:1:2:2 to about 1:1:10:10 (w / w).

[0455] Any of the embodiments described herein may comprise a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in an mRNA ratio expressed in micrograms (μg).

[0456] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in a ratio (w / w) of approximately 32 micrograms of the first mRNA to approximately 32 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA.

[0457] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in a ratio (w / w) of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA.

[0458] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in a ratio (w / w) of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 96 micrograms of the third mRNA to approximately 96 micrograms of the fourth mRNA.

[0459] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in a ratio (w / w) of 32 micrograms of the first mRNA to 32 micrograms of the second mRNA to 64 micrograms of the third mRNA to 64 micrograms of the fourth mRNA.

[0460] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in a ratio (w / w) of 16 micrograms of the first mRNA to 16 micrograms of the second mRNA to 64 micrograms of the third mRNA to 64 micrograms of the fourth mRNA.

[0461] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in a ratio (w / w) of 16 micrograms of the first mRNA to 16 micrograms of the second mRNA to 96 micrograms of the third mRNA to 96 micrograms of the fourth mRNA.

[0462] Any of the embodiments described herein may contain a total of 130 micrograms of mRNA.

[0463] Any of the embodiments described herein may contain a total of 160 micrograms of mRNA.

[0464] Any of the embodiments described herein may contain a total of 200 micrograms of mRNA.

[0465] Any of the embodiments described herein may contain a total of 224 micrograms of mRNA.

[0466] Any of the embodiments described herein may contain a total of 130 to 224 micrograms of mRNA.

[0467] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:2 (e.g., μg).

[0468] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:3 (e.g., μg).

[0469] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:4 (e.g., μg).

[0470] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:5 (e.g., μg).

[0471] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:6 (e.g., μg).

[0472] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:7 (e.g., μg).

[0473] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:8 (e.g., μg).

[0474] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:9 (e.g., μg).

[0475] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of approximately 1:1:10 (e.g., μg).

[0476] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:2 (e.g., μg).

[0477] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:3 (e.g., μg).

[0478] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus, in an mRNA ratio of 1:1:4 (e.g., μg).

[0479] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:5 (e.g., μg).

[0480] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:6 (e.g., μg).

[0481] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:7 (e.g., μg).

[0482] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:8 (e.g., μg).

[0483] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:9 (e.g., μg).

[0484] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), and the HA antigen of an influenza B virus in an mRNA ratio of 1:1:10 (e.g., μg).

[0485] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:2 (e.g., μg).

[0486] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:3 (e.g., μg).

[0487] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:4 (e.g., μg).

[0488] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:5 (e.g., μg).

[0489] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:6 (e.g., μg).

[0490] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:7 (e.g., μg).

[0491] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:8 (e.g., μg).

[0492] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:9 (e.g., μg).

[0493] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of approximately 1:1:10 (e.g., μg).

[0494] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:2 (e.g., μg).

[0495] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:3 (e.g., μg).

[0496] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:4 (e.g., μg).

[0497] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:5 (e.g., μg).

[0498] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:6 (e.g., μg).

[0499] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:7 (e.g., μg).

[0500] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:8 (e.g., μg).

[0501] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:9 (e.g., μg).

[0502] Any of the embodiments described herein may contain the first mRNA, the second mRNA, and the third mRNA in a ratio of 1:1:10 (e.g., μg).

[0503] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:2:2 (e.g., μg).

[0504] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:3:3 (e.g., μg).

[0505] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:4:4 (e.g., μg).

[0506] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:5:5 (e.g., μg).

[0507] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:6:6 (e.g., μg).

[0508] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:7:7 (e.g., μg).

[0509] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:8:8 (e.g., μg).

[0510] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:9:9 (e.g., μg).

[0511] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of approximately 1:1:10:10 (e.g., μg).

[0512] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:2:2 (e.g., μg).

[0513] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:3:3 (e.g., μg).

[0514] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:4:4 (e.g., μg).

[0515] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:5:5 (e.g., μg).

[0516] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:6:6 (e.g., μg).

[0517] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:7:7 (e.g., μg).

[0518] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:8:8 (e.g., μg).

[0519] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:9:9 (e.g., μg).

[0520] A composition of any embodiment described herein may contain the HA antigen of a first influenza A virus, the HA antigen of a second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes), the HA antigen of a first influenza B virus, and the HA antigen of a second influenza B virus (where the first influenza B virus and the second influenza B virus are different lineages) in an mRNA ratio of 1:1:10:10 (e.g., μg).

[0521] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:2:2 (e.g., μg).

[0522] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:3:3 (e.g., μg).

[0523] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:4:4 (e.g., μg).

[0524] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:5:5 (e.g., μg).

[0525] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:6:6 (e.g., μg).

[0526] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:7:7 (e.g., μg).

[0527] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:8:8 (e.g., μg).

[0528] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:9:9 (e.g., μg).

[0529] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of approximately 1:1:10:10 (e.g., μg).

[0530] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:2:2 (e.g., μg).

[0531] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:3:3 (e.g., μg).

[0532] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:4:4 (e.g., μg).

[0533] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:5:5 (e.g., μg).

[0534] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:6:6 (e.g., μg).

[0535] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:7:7 (e.g., μg).

[0536] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:8:8 (e.g., μg).

[0537] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:9:9 (e.g., μg).

[0538] Any of the embodiments described herein may contain the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio of 1:1:10:10 (e.g., μg).

[0539] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in the ratio of approximately 32 micrograms of the first mRNA to approximately 32 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA.

[0540] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in the ratio of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA.

[0541] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in the ratio of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 96 micrograms of the third mRNA to approximately 96 micrograms of the fourth mRNA.

[0542] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in the ratio of 32 micrograms of the first mRNA to 32 micrograms of the second mRNA to 64 micrograms of the third mRNA to 64 micrograms of the fourth mRNA.

[0543] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in the ratio of 16 micrograms of the first mRNA to 16 micrograms of the second mRNA to 64 micrograms of the third mRNA to 64 micrograms of the fourth mRNA.

[0544] A composition of any embodiment described herein may contain a first mRNA, a second mRNA, a third mRNA, and a fourth mRNA in the ratio of 16 micrograms of the first mRNA to 16 micrograms of the second mRNA to 96 micrograms of the third mRNA to 96 micrograms of the fourth mRNA.

[0545] Any of the embodiments described herein may contain a total of 130 micrograms of mRNA.

[0546] Any of the embodiments described herein may contain a total of 160 micrograms of mRNA.

[0547] Any of the embodiments described herein may contain a total of 200 micrograms of mRNA.

[0548] Any of the embodiments described herein may contain a total of 224 micrograms of mRNA.

[0549] Any of the embodiments described herein may contain a total of 130 to 224 micrograms of mRNA.

[0550] Any of the compositions of the embodiments described herein may include ratios expressed, for example, in picograms (pg), nanograms (ng), micrograms (μg), milligrams (mg), etc.

[0551] IV. Lipid Nanoparticles (LNPs) The LNPs of this disclosure include four categories of lipids: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids; and (iv) helper lipids.

[0552] A. Cationic lipids Ionizable lipids facilitate mRNA encapsulation and may be cationic lipids. Cationic lipids provide a positively charged environment at low pH, facilitating the efficient encapsulation of negatively charged mRNA drug substances. Exemplary cationic lipids are shown in Table 1 below.

[0553] [Table 1]

[0554] [Table 2]

[0555] [Table 3]

[0556] [Table 4]

[0557] Cationic lipids include [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-nona-2-en-1-yl)9-((4 -(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadeca-9-enoyl]oxypropyl](Z)-octadeca-9-enoate( DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-C hol); Tetrakis(8-methylnonyl)3,3',3",3”'-(((methylazandiyl)bis(propane-3,1diyl))bis(azantriyl))tetrapropionate(306Oi10); Decyl(2-(dioctylammonio)ethyl)phosphate(9A1P9); Ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate(A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azandiyl)dipropionate (BAME-O16B); 1,1'-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane- 2-ol)(C12-200); 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione(cKK-E12); hexa(octan-3-yl)9,9',9”,9”',9””,9””'-((((benzene-1,3,5-tricarbonyl)iris(azandiyl))tris(propane-3,1-diyl))tris(azantriyl))hexanonaate(FTT5);(((3, 6-Dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)bis(axantriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9”Z,9”'Z,12Z,12'Z,12”Z,12”'Z)-tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin);TT3;N1,N3,N5-Tris(3-(didodecylamino)propyl)benzene-1,3,5-tricalboxate Mido; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 5); IM-001; and combinations thereof may be selected from the group.

[0558] In certain embodiments, cationic lipids are biodegradable.

[0559] In certain embodiments, cationic lipids are not biodegradable.

[0560] In certain embodiments, cationic lipids are cleavable.

[0561] In certain embodiments, cationic lipids are not cleavable.

[0562] Cationic lipids are described in more detail in Dong et al. (PNAS.111;11:3955-60.2014); Fenton et al. (Adv.Mater.28:2939.2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference.

[0563] B. PEGylated lipids PEGylated lipid components provide control over the particle size and stability of nanoparticles. Adding such components can prevent complex aggregation, extend circulating life, and provide a means to increase the delivery of lipid-nucleic acid drug compositions to target tissues (Klibanov et al., FEBS Letters 268(1):235-7. 1990). These components may be selected to be rapidly replaced from the drug composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0564] Examples of PEGylated lipids include, but are not limited to, polyethylene glycol (PEG) with a maximum length of 5 kDa covalently bonded to a lipid having an alkyl chain of C6-C20 length (e.g., C8, C10, C12, C14, C16, or C18), such as derivatized ceramide (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipids are 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkyloxypropyl carbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0565] In certain embodiments, PEG has a high molecular weight, for example, 2000 to 2400 g / mol. In certain embodiments, PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipids of this specification are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipids of this specification are DMG-PEG2000.

[0566] C. Cholesterol lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP contains one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamide cholesterol) and 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al.). al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol ester ("ICE"; International Publication No. 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigma-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24-cholesterol); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24, Examples include 25-dihydrolanosterol; thymosterol (5α-cholesta-8,24-diene-3β-ol); lasosterol (5α-cholesta-7-ene-3β-ol); diosgenin ((3β,25R)-spirosto-5-ene-3-ol); campesterol (campesto-5-ene-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol); cholesteryl margallate (cholesta-5-ene-3β-ylheptadecanoate); cholesteryl oleate; cholesteryl stearate; and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in LNPs is cholesterol.

[0567] D. Helper lipids Helper lipids enhance the structural stability of LNPs and assist in LNP extrusion into endosomes. This improves the uptake and release of mRNA drug payloads. In some embodiments, the helper lipids are zwitterionic lipids with fusion properties that enhance the uptake and release of drug payloads. Examples of helper lipids include 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dieridoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0568] Other exemplary helper lipids include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramides, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0569] In various embodiments, the LNP comprises (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0570] E. Molar ratio of lipid components The molar ratios of the above components are important for the effectiveness of LNPs in mRNA delivery. The molar ratio of cationic lipids, PEGylated lipids, cholesterol lipids, and helper lipids is A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipids in LNPs to total lipids (i.e., A) is 35-55%, for example 35-50% (e.g., 38-42%, for example 40%, or 45-50%). In some embodiments, the molar ratio of PEGylated lipid components to total lipids (i.e., B) is 0.25-2.75% (e.g., 1-2%, for example 1.5%). In some embodiments, the molar ratio of cholesterol lipids to total lipids (i.e., C) is 20-50% (e.g., 27-30%, for example 28.5%, or 38-43%). In some embodiments, the molar ratio of helper lipids to total lipids (i.e., D) is 5–35% (e.g., 28–32%, e.g., 30%, or 8–12%, e.g., 10%). In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the helper lipids. In some embodiments, the molar ratio of cationic lipids to helper lipids in LNP is greater than 1.

[0571] In certain embodiments, the LNP of this disclosure is Cationic lipids in molar ratios of 35% to 55% or 40% to 50% (for example, cationic lipids in 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%); Polyethylene glycol (PEG) conjugate (PEG-conjugated) lipids in molar ratios of 0.25% to 2.75% or 1.00% to 2.00% (e.g., PEG-conjugated 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%); Cholesterol-based lipids in molar ratios of 20%~45%, 20%~50%, 25%~45%, or 28.5%~43% (e.g., cholesterol-based lipids in 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 Contains helper lipids in molar ratios of 5% to 35%, 8% to 30%, or 10% to 30% (for example, helper lipids in molar ratios 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%). (Here, all molar ratios are relative to the total lipid content of LNP.)

[0572] In certain embodiments, the LNP comprises a cationic lipid in a molar ratio of 40%, a PEGylated lipid in a molar ratio of 1.5%, a cholesterol-based lipid in a molar ratio of 28.5%, and a helper lipid in a molar ratio of 30%.

[0573] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0574] In various embodiments, the cholesterol-based lipid is cholesterol.

[0575] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0576] In certain embodiments, the LNP comprises OF-O2 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%.

[0577] In certain 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%.

[0578] In certain 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%.

[0579] In certain 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%.

[0580] In certain 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%.

[0581] In certain 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%.

[0582] In certain 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%.

[0583] In certain embodiments, the LNP comprises OF-O2 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as “Lipid A”.

[0584] In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as “Lipid B”.

[0585] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 in a 40% molar ratio; DMG-PEG2000 in a 1.5% molar ratio; cholesterol in a 28.5% molar ratio; and DOPE in a 30% molar ratio. This LNP formulation is referred to herein as "Lipid C".

[0586] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid D".

[0587] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 40%; DMG-PEG2000 in a molar ratio of 1.5%; cholesterol in a molar ratio of 28.5%; and DOPE in a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid E".

[0588] In certain embodiments, the LNP comprises 50% molar ratio of 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 38.5% molar ratio of cholesterol; and 1.5% molar ratio of 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0589] In certain embodiments, the LNP comprises (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 9.4%; cholesterol in a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.6%.

[0590] In certain embodiments, the LNP comprises (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 10%; cholesterol in a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.7%.

[0591] In certain embodiments, the LNP comprises IM-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0592] To calculate the actual amount of each lipid contained in an LNP formulation, first determine the molar amount of the cationic lipid 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 transported by the LNP). Next, calculate the molar amount of each of the other lipids based on the molar amount of the cationic lipid and the selected molar ratio. Then, convert these molar amounts to weight using the molecular weight of each lipid.

[0593] F. Buffering agents and other components To stabilize nucleic acids and / or LNPs (e.g., to extend the shelf life of a vaccine product), to facilitate the administration of LNP pharmaceutical compositions, and / or to improve the in vivo expression of nucleic acids, nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeted ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients include parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0594] The LNP compositions of this disclosure may be provided in liquid or lyophilized form. Various cryoprotective agents may be used, but are not limited to sucrose, trehalose, glucose, mannitol, mannose, and dextrose. The cryoprotective agent may constitute 5 to 30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, 5 to 30% (e.g., 10%) (w / v). Once formulated with the cryoprotective agent, the LNP composition may be frozen (or lyophilized and cryopreserved) at -20°C to -80°C.

[0595] The LNP composition may be provided to the patient in a buffered aqueous solution (which can be thawed if pre-frozen, or reconstituted with a buffered aqueous solution at the bedside if pre-lyophilized). The buffered solution is preferably isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffered solution is phosphate-buffered saline (PBS).

[0596] Process for producing V.LNP vaccine These LNPs can be prepared by various techniques currently known in the art. For example, multilayer vesicles (MLVs) can be prepared according to the prior art, for example, by dissolving the lipids in a suitable solvent to deposit the selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. Then, an aqueous phase can be added to the vessel by a vortex motion that results in the formation of the MLV. Then, single-layer vesicles (ULVs) can be formed by homogenizing, sonicating, or extruding the multilayer vesicles. Furthermore, single-layer vesicles can be formed by detergent removal techniques.

[0597] Various methods are described in U.S. Patent Publication No. 2011 / 0244026, U.S. Patent Publication No. 2016 / 0038432, U.S. Patent Publication No. 2018 / 0153822, U.S. Patent Publication No. 2018 / 0125989, and U.S. Patent Publication No. 2021 / 0046192, which can be used to produce LNP vaccines. One exemplary process involves encapsulating mRNA by mixing it with a mixture of lipids without first pre-forming the lipids into lipid nanoparticles, as described in U.S. Patent Publication No. 2016 / 0038432. Another exemplary process involves encapsulating mRNA by mixing it with pre-formed LNPs, as described in U.S. Patent Publication No. 2018 / 0153822.

[0598] In some embodiments, the process for preparing mRNA-loaded LNPs includes heating one or more solutions to a temperature higher than ambient temperature, wherein the one or more solutions are a solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing mRNA encapsulated in the LNP. In some embodiments, the process includes heating one or both of the mRNA solution and the pre-formed LNP solution before the mixing step. In some embodiments, the process includes heating one or more of the solutions containing the pre-formed LNP, the mRNA solution, and the mRNA encapsulated in the LNP during the mixing step. In some embodiments, the process includes heating the mRNA encapsulated in the LNP after the mixing step. In some embodiments, the temperature at which one or more of the solutions are heated is approximately 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature at which one or more of the solutions are heated is in the range of about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C. In some embodiments, the temperature is about 65°C.

[0599] Various methods can be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA may be dissolved directly in the buffer solution described herein. In some embodiments, the mRNA solution may be prepared by mixing the mRNA stock solution with the buffer solution before mixing it with the lipid solution for mounting. In some embodiments, the mRNA solution may be prepared by mixing the mRNA stock solution with the buffer solution immediately before mixing it with the lipid solution for mounting. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or buffer solution at concentrations of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or higher.

[0600] In some embodiments, the mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a higher flow rate than the mRNA stock solution. For example, the buffer solution may be mixed at a flow rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times that of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100 to 6000 ml / min (e.g., about 100 to 300 ml / min, 300 to 600 ml / min, 600 to 1200 ml / min, 1200 to 2400 ml / min, 2400 to 3600 ml / min, 3600 to 4800 ml / min, 4800 to 6000 ml / min, or 60 to 420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of approximately 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or more.

[0601] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10 to 600 ml / min (for example, approximately 5 to 50 ml / min, approximately 10 to 30 ml / min, approximately 30 to 60 ml / min, approximately 60 to 120 ml / min, approximately 120 to 240 ml / min, approximately 240 to 360 ml / min, approximately 360 to 480 ml / min, or approximately 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of approximately 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or more.

[0602] The process of incorporating desired mRNA into lipid nanoparticles is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. Nucleic acids incorporated into LNPs may be located entirely or partially within the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the outer surface of the lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as “encapsulation,” in which the nucleic acid is contained entirely or substantially within the internal space of the lipid nanoparticle.

[0603] Suitable LNPs can be produced in a variety of sizes. In some embodiments, reducing the size of lipid nanoparticles is associated with more efficient mRNA delivery. The selection of an appropriate LNP size can take into account to some extent the site of the target cell or tissue and the intended use for which the lipid nanoparticles are produced.

[0604] Various methods known in the art can be used for sizing a population of lipid nanoparticles. In a preferred method herein, LNP particle size is measured using a Zetasizer Nano ZS (Malvern Panalytical). In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is placed in a cuvette and then in a Zetasizer machine. The z-mean diameter (nm) or cumulant mean is considered the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average diameter of the formed LNPs can be reduced by sonication. Intermittent sonication cycles alternating with quasi-elastic light scattering (QELS) evaluation can lead to efficient lipid nanoparticle synthesis.

[0605] In some embodiments, the majority of the purified LNPs, i.e., more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of approximately 70–150 nm (e.g., approximately 145 nm, approximately 140 nm, approximately 135 nm, approximately 130 nm, approximately 125 nm, approximately 120 nm, approximately 115 nm, approximately 110 nm, approximately 105 nm, approximately 100 nm, approximately 95 nm, approximately 90 nm, approximately 85 nm, or approximately 80 nm). In some embodiments, substantially all (e.g., more than 80 or 90%) of the purified lipid nanoparticles have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0606] In certain embodiments, the LNP has an average diameter of 30 to 200 nm.

[0607] In various embodiments, the LNP has an average diameter of 80 to 150 nm.

[0608] In some embodiments, the LNPs in the composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0609] In some embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the LNPs in the composition have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm) or about 50-70 nm (e.g., about 55-65 nm), which is particularly suitable for lung delivery by spraying.

[0610] In some embodiments, the degree of dispersion of LNPs in the pharmaceutical compositions provided herein, or the measure of molecular size heterogeneity (PDI), is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI can be measured by a Zetasizer machine as described above.

[0611] In some embodiments, about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., 80% or more than 90%) of the purified lipid nanoparticles in the pharmaceutical compositions encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99%; or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or more than 99%. Typically, the lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%).

[0612] In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, the typical LNPs of this specification have an N / P ratio of 4.

[0613] In some embodiments, the pharmaceutical compositions according to this disclosure contain 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 compositions contain 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.

[0614] In some embodiments, mRNA may be produced by chemical synthesis or by in vitro transcription (IVT) of a DNA template. An exemplary process for producing and purifying mRNA is described in Example 1. In this process, the IVT process, mRNA transcripts are produced using a cDNA template, which is then degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is purified again by deep filtration and TFF.

[0615] Next, mRNA is prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of LNP. The amphiphilic solution for dissolving the four lipid components of LNP may be an alcoholic solution. In some embodiments, the alcohol is ethanol. The aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0 to 7.0, for example, 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 salts, potassium salts, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0616] An exemplary and non-limiting process for preparing mRNA-LNP compositions involves mixing a buffered mRNA solution with a lipid solution in ethanol in a controlled and homogeneous manner, where the lipid:mRNA ratio is maintained throughout the mixing process. In this exemplary example, mRNA is present 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 lipids, and helper lipids) is dissolved in ethanol. The mRNA aqueous solution and the ethanol lipid solution are mixed in a volume ratio of 4:1 in a "T" mixer equipped with a nearly "pulsation-free" pump system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. Using a TFF, the nascent LNPs obtained immediately after formation by the T mixing process can be concentrated and buffer exchanged. The diafiltration process is a continuous operation in which the volume is kept constant by adding an appropriate buffer solution at the same flow rate as the permeate flow.

[0617] VI. Packaging and Use of mRNA-LNP Vaccines 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 may be in the form of an immediate formulation in which the LNP composition is lyophilized and reconstituted with physiological buffer (e.g., PBS) immediately before use. The vaccine composition may also be shipped and supplied in the form of an aqueous solution or frozen aqueous solution and can be administered directly to the subject without reconstitution (after thawing if previously frozen).

[0618] Accordingly, this disclosure provides products such as kits that provide an mRNA-LNP vaccine in a single container, or provide an mRNA-LNP vaccine in one container (e.g., a first container) and physiological buffers for reconstitution in another container (e.g., a second container). The containers may contain single-use doses or multi-use doses. The containers may be pre-treated glass vials or ampoules. The products may also include instructions for use.

[0619] In certain embodiments, the mRNA-LNP vaccine is provided for use by intramuscular (IM) injection. The vaccine can be injected into a target, for example, in the deltoid muscle of the upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber type). In some embodiments, the vaccine is provided for use by inhalation and is provided in a pre-filled pump, aerosol generator or inhaler.

[0620] mRNA-LNP vaccines can be administered to those in need in a prophylactic dose, i.e., a dose that provides sufficient immune protection against the target pathogen for a sufficient period of time (e.g., 1 year, 2 years, 5 years, 10 years, or a lifetime). Sufficient immune protection may, for example, prevent or alleviate symptoms associated with infection by the pathogen. In some embodiments, multiple doses of the vaccine (e.g., two doses) are administered (e.g., by injection) to those in need to achieve the desired prophylactic effect. The doses (e.g., initial dose and bolus dose) may be divided at intervals of at least, for example, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, or 10 years.

[0621] VII. Vector In one embodiment, a vector comprising an mRNA composition disclosed herein is disclosed herein. An RNA sequence encoding a protein of interest (e.g., mRNA encoding the influenza HA protein) can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors for specific purposes include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0622] In certain embodiments, a vector can be used to express mRNA in host cells. In various embodiments, the vector can be used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0623] In some embodiments, the vectors disclosed herein may comprise at least the following from 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding the 5'UTR; a polynucleotide sequence encoding the ORF; a polynucleotide sequence encoding the 3'UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may comprise a poly(A) sequence and / or a polynucleotide sequence encoding a polyadenylation signal.

[0624] Various RNA polymerase promoters are known. In some embodiments, the promoter may be the T7 RNA polymerase promoter. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known.

[0625] Host cells (e.g., mammalian cells, e.g., human cells) containing the vector or RNA composition disclosed herein are also disclosed herein.

[0626] Polynucleotides can be introduced into target cells using any of the many different methods, including, but not limited to, commercially available methods such as electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser I (BioRad, Denver, Cologne.), multiporators (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, "gene guns" (biological particle delivery systems such as Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70), or TransIT-RNA transfection kits (Mirus, Madison, WI).

[0627] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is liposomes (e.g., artificial membrane vesicles).

[0628] Various assays may be performed to confirm the presence of mRNA sequences in host cells, regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this disclosure.

[0629] VIII. Self-replicating RNA and trans-replicating RNA Self-replicating RNA: In one embodiment, a self-replicating RNA encoding an influenza protein (e.g., influenza HA protein) is disclosed herein.

[0630] Self-replicating RNA can be produced, for example, by using replication elements derived from an alphavirus and substituting the structural viral protein with a nucleotide sequence encoding the target protein (e.g., influenza HA protein). Self-replicating RNA is typically a positive-chain molecule that can be directly translated after being delivered to a cell, and this translation provides RNA-dependent RNA polymerase, which 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 and collinear subgenomic transcripts can be translated themselves to provide in-situ expression of the encoded antigen (i.e., influenza HA protein antigen), or they can be transcribed to provide further transcripts with the same sense as the delivered RNA that is translated to provide in-situ expression of the antigen. The overall result of this series of transcriptions is that many introduced replicon RNAs are widely amplified, so that the encoded antigen becomes the cell's primary polypeptide product.

[0631] One preferred system for achieving self-replication in this manner is the use of alphavirus-based replicons. These replicons are positive-sense RNAs that, after delivery to a cell, result in the translation of a replicase (or replicase transcriptase). The replicase is translated into a polyprotein that self-cleaves to provide a replication complex, thereby generating a genomic copy of the delivered positive-sense RNA. These negative-sense transcripts can themselves be transcribed to give further copies of the positive-sense parental RNA, and can also give subgenomic transcripts encoding antigens. Thus, translation of the subgenomic transcripts results in in-situ expression of the antigen by the infected cell. Suitable alphavirus replicons can be replicases derived from Sindbis virus, Semlik Forest virus, Eastern Equine Encephalitis virus, Venezuelan Encephalitis virus, etc. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 variant of VEEV has been used in replicons. See the following references: International Publication No. 2005 / 113782 (incorporated herein by reference).

[0632] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an influenza HA protein antigen. The polymerase may be, for example, an alphaviral replicase containing one or more alphaviral proteins such as nsP1, nsP2, nsP3, and nsP4. While the natural alphaviral genome encodes structural virion proteins in addition to non-structural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphaviral structural proteins. Therefore, the self-replicating RNA may result in the production of its own genomic RNA copies in 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 persist on its own in the infectious form. The alphaviral structural proteins necessary for persistence in wild-type virus are not present in the self-replicating RNA of this disclosure, and their positions are occupied by genes encoding the immunogen of interest; therefore, the subgenome transcript encodes its immunogen, not the structural alphaviral virion protein. Self-replicating RNA is described in more detail in International Publication No. 2011 / 005799 (incorporated herein by reference).

[0633] Trans-replicated RNA: In one embodiment, a trans-replicating RNA encoding an influenza protein is disclosed herein.

[0634] Trans-replicating RNA has similar elements to the self-replicating RNA described above. However, trans-replicating RNA uses two distinct RNA molecules. The first RNA molecule encodes the RNA replicase described above (e.g., alphavirus replicase), and the second RNA molecule encodes the target protein (e.g., influenza protein antigen). The RNA replicase replicates 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 described in more detail in International Publication No. 2017 / 162265 (incorporated herein by reference).

[0635] IX. Pharmaceutical Compositions RNA purified in accordance with this disclosure may be useful, for example, as a component in a pharmaceutical composition for use as a vaccine. These compositions typically comprise RNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions of this disclosure may also comprise one or more further components, such as small molecule immunostimulants (e.g., TLR agonists). The pharmaceutical compositions of this disclosure may also comprise 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 certain embodiments, the composition comprises antigen-coding nucleic acid molecules encapsulated within the LNPs.

[0636] X. Method of eviction The influenza mRNA vaccine compositions disclosed herein can be administered to subjects to induce an immune response to one or more influenza proteins, and the anti-antigen antibody titer of the subjects increases after vaccination compared to the anti-antigen antibody titer of subjects not vaccinated with the influenza mRNA vaccine compositions disclosed herein or compared to an alternative vaccine against influenza. "Anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0637] In one embodiment, the Disclosure provides a method for inducing an immune response to influenza or protecting a subject from influenza infection, comprising administering the influenza mRNA vaccine composition described herein to the subject. In another embodiment, the Disclosure provides a composition for use in inducing an immune response to influenza or protecting a subject from influenza infection by administering the above-described influenza mRNA vaccine composition to the subject. The Disclosure also provides the influenza mRNA vaccine composition described herein for use in the manufacture of a vaccine for inducing an immune response to influenza or a vaccine for protecting a subject from influenza infection.

[0638] In one embodiment, the Disclosure provides a method for inducing an immune response to influenza type A or protecting a subject from influenza type A infection by administering the influenza mRNA vaccine composition described herein to the subject. In another embodiment, the Disclosure provides a method for inducing an immune response to influenza type B or protecting a subject from influenza type B infection by administering the influenza mRNA vaccine composition described herein to the subject.

[0639] In one embodiment, the Disclosure provides compositions disclosed herein for use in a method of inducing an immune response to influenza A or protecting a subject from influenza A infection. In another embodiment, the Disclosure provides compositions disclosed herein for use in a method of inducing an immune response to influenza B or protecting a subject from influenza B infection.

[0640] This disclosure also provides the influenza mRNA vaccine composition described herein for use in the manufacture of a pharmaceutical for inducing an immune response to influenza A or a pharmaceutical for protecting a subject from influenza A infection. This disclosure further provides the influenza mRNA vaccine composition described herein for use in the manufacture of a pharmaceutical for inducing an immune response to influenza B or a pharmaceutical for protecting a subject from influenza B infection.

[0641] In another embodiment, the present invention provides a method for immunizing a subject against one or more influenza viruses. The present invention further provides a method for inducing an immune response in a subject against one or more influenza viruses. In some embodiments, the method comprises administering an effective amount of the influenza mRNA vaccine composition described herein to the subject.

[0642] In certain embodiments, a subject has the same or higher serum concentration of neutralizing antibodies against influenza A after administration of the influenza mRNA vaccine composition described herein, compared to a subject administered with a single antigen composition containing mRNA encoding the influenza protein. In certain embodiments, a subject has the same or higher serum concentration of neutralizing antibodies against influenza B after administration of the influenza mRNA vaccine composition described herein, compared to a subject administered with a single antigen composition containing mRNA encoding the influenza protein.

[0643] In certain embodiments, subjects exhibit equivalent serum concentrations of neutralizing antibodies against influenza A after administration of the influenza mRNA vaccine composition described herein, compared to subjects administered with a protein-type A influenza vaccine. In certain embodiments, subjects exhibit equivalent serum concentrations of neutralizing antibodies against influenza B after administration of the influenza mRNA vaccine composition described herein, compared to subjects administered with a protein-type B influenza vaccine.

[0644] In certain embodiments, the influenza mRNA vaccine composition described herein increases the serum concentration of neutralizing antibodies against influenza A in a subject. In certain embodiments, the influenza mRNA vaccine composition described herein increases the serum concentration of neutralizing antibodies against influenza B in a subject.

[0645] In certain embodiments, the influenza mRNA vaccine composition described herein increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in the subject. In certain embodiments, the influenza mRNA vaccine composition described herein increases the serum concentration of neutralizing antibodies against influenza B Yamagata lineage and / or influenza B Victoria lineage in the subject.

[0646] In certain embodiments, the compositions described herein increase the serum concentration of neutralizing antibodies in subjects with pre-existing influenza immunity.

[0647] In various embodiments, the immunization methods provided herein induce a broad protective immune response against multiple epitopes within one or more influenza viruses. In various embodiments, the immunization methods provided herein induce a broad neutralizing immune response against one or more influenza viruses. In some embodiments, the immune response includes an antibody response. Thus, in various embodiments, the influenza mRNA vaccine compositions described herein may provide broad cross-protection against various types of influenza viruses. In some embodiments, the influenza mRNA vaccine compositions described herein provide cross-protection against avian, porcine, seasonal, and / or pandemic influenza viruses. In some embodiments, the influenza mRNA vaccine compositions described herein provide cross-protection against one or more subtypes of influenza A, B, or C. In some embodiments, the influenza mRNA vaccine compositions described herein provide cross-protection against multiple strains of influenza A H1 subtype virus (e.g., H1N1), influenza A H3 subtype virus (e.g., H3N2), influenza A H5 subtype virus (e.g., H5N1), and / or influenza B virus (e.g., Yamagata lineage, Victoria lineage).

[0648] In some embodiments, the methods of the present invention can induce an improved immune response to one or more seasonal influenza strains. Exemplary seasonal strains include, but are not limited to, A / Puerto Rico / 8 / 1934, A / FortMonmouth / 1 / 1947, A / Chile / 1 / 1983, A / Texas / 36 / 1991, A / Singapore / 6 / 1986, A / Beijing / 32 / 1992, A / NewCaledonia / 20 / 1999, and A / Solomon. Islands / 03 / 2006, A / Brisbane / 59 / 2007, A(H3N2) virus antigenically similar to cell proliferation prototype virus A / Victoria / 361 / 2011, A / Beijing / 262 / 95(H1N1)-like virus, A / Brisbane / 02 / 2018(H1N1)pdm09-like virus, A / Brisbane / 10 / 2007(H3N2)-like virus, A / California / 7 / 2004(H3N2)-like virus, A / California / 7 / 2009(H1N1)-like virus, A / California / 7 / 2009(H1N1)pdm09-like virus, A / Cambodia / e0826360 / 2020(H3N2) virus, A / Darwin / 6 / 2021(H3N2) virus, A / Fujian / 411 / 2002(H3N2) virus, A / Fujian / 411 / 2002(H3N2) virus, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09- virus, A / Hawaii / 70 / 2019(H1N1)pdm09- virus, A / Hong Kong / 2671 / 2019(H3N2)- virus, A / Hong Kong / 45 / 2019(H3N2)- virus, A / Hong Kong / 4801 / 2014(H3N2)-like virus, A / Kansas / 14 / 2017(H3N2)-like virus, A / Michigan / 45 / 2015(H1N1)pdm09-like virus, A / Moscow / 10 / 99(H3N2)-like virus, A / NewCaledonia / 20 / 99(H1N1)-like virus, A / Perth / 16 / 2009(H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2)-like virus, A / SolomonIslands / 3 / 2006(H1N1)-type virus, A / SouthAustralia / 34 / 2019(H3N2)-type virus, A / Switzerland / 8060 / 2017(H3N2)-type virus, A / Switzerland / 9715293 / 2013(H3N2)-type virus, A / Sydney / 5 / 97(H3N2)-type virus, A / Texas / 50 / 2012(H3N2)-type virus, A / Victoria / 2570 / 2019(H1N1)pdm09-type virus, A / Victoria / 2570 / 2019(H1N1)pdm09-type virus, A / Victoria / 361 / 2011(H3N2)-type virus A / Wellington / 1 / 2004(H3N2)-like virus, A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Wisconsin / 67 / 2005(H3N2)-like virus, B / Austria / 1359417 / 20121(B / Victoria lineage)-like virus, B / Beijing / 184 / 93-like virus, B / Brisbane / 60 / 2008-like virus, B / Colorado / 06 / 2017-like virus (B / Victoria / 2 / 87 lineage), B / Florida / 4 / 2006-like virus, B / HongKong / 330 / 2001- virus, B / Malaysia / 2506 / 2004- virus, B / Massachusetts / 2 / 2012- virus, B / Phuket / 3073 / 2013 (B / Yamagata lineage)- virus, B / Phuket / 3073 / 2013- virus, B / Phuket / 3073 / 2013- virus (B / Yamagata / 16 / 88 lineage), B / Shangdong / 7 / 97- virus, B Examples include the B / Shanghai / 361 / 2002-like virus, the B / Sichuan / 379 / 99-like virus, the B / Washington / 02 / 2019 (B / Victoria lineage)-like virus, the B / Washington / 02 / 2019-like (B / Victoria lineage) virus, the A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, and the B / Wisconsin / 1 / 2010-like virus. In some embodiments, the methods of the present invention can induce an improved immune response to one or more pandemic influenza strains. Exemplary pandemic strains include, but are not limited to, A / California / 07 / 2009, A / California / 04 / 2009, A / Belgium / 145 / 2009, A / South Carolina / 01 / 1918, and A / New Jersey / 1976. Examples of pandemic subtypes include, in particular, H1N1, H5N1, H2N2, H3N2, H9N2, H7N7, H7N3, H7N9, and H10N7 subtypes. In some embodiments, the methods of the present invention can induce an improved immune response to one or more swine influenza strains. Exemplary swine strains include, but are not limited to, the A / NJEJERS / 1976 isolate and A / California / 07 / 2009. In some embodiments, the methods of the present invention can induce an improved immune response to one or more avian influenza strains. Exemplary avian strains include, but are not limited to, H5N1, H7N3, H7N7, H7N9, and H9N2. Additional pandemic, seasonal, avian, and / or swine influenza strains are known in the art.

[0649] In some embodiments, the present invention provides a method for preventing or treating influenza infection by administering an influenza mRNA vaccine composition described herein to a subject in need thereof. In some embodiments, the subject is infected with or susceptible to influenza infection. In some embodiments, the subject is considered infected with influenza infection if the subject exhibits one or more symptoms generally associated with influenza infection. In some embodiments, the subject is known to be exposed to or is thought to be exposed to the influenza virus. In some embodiments, the subject is considered susceptible to influenza infection if the subject is known to be exposed to or is thought to be exposed to the influenza virus. In some embodiments, the subject is known to be exposed to or is thought to be exposed to the influenza virus if the subject has been in contact with other individuals known to be infected with or suspected to be infected with the influenza virus, and / or the subject is present in or has been present in an area where an influenza outbreak is known to be or is thought to be occurring.

[0650] In various embodiments, the influenza mRNA vaccine compositions described herein may be administered before or after the onset of one or more symptoms of influenza infection. In some embodiments, the influenza mRNA vaccine compositions described herein are administered as prophylactic agents. In such embodiments, the methods of the present invention are effective in preventing or protecting subjects from influenza virus infection. In some embodiments, the influenza mRNA vaccine compositions of the present invention are used as components of seasonal and / or pandemic influenza vaccines, or as part of an influenza vaccination regimen intended to provide long-term (multi-season) protection. In some embodiments, the influenza mRNA vaccine compositions described herein are used to treat symptoms of influenza infection.

[0651] In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is livestock or pets (e.g., dogs, cats, sheep, cattle, and / or pigs). In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a bird (e.g., a chicken).

[0652] In some embodiments, the subject is human. In certain embodiments, the subject is adult, adolescent, or infant. In some embodiments, the human subject is less than 6 months old. In some embodiments, the human subject is 6 months or older, 6 months to 35 months old, 36 months to 8 years old, or 9 years or older. In some embodiments, the human subject is 55 years or older, for example, 60 years or older, or 65 years or older. Administration of the composition and / or implementation of intrauterine treatment are also intended by the present invention.

[0653] The present invention is further defined by reference to the following numbered paragraphs: 1. A composition comprising at least three messenger RNAs (mRNAs), wherein at least three mRNAs are (i) The first mRNA encoding the HA antigen of the first influenza A virus; (ii) A second mRNA encoding the HA antigen of the second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); (iii) A third mRNA encoding the HA antigen of the first influenza B virus. It includes an open reading frame (ORF) encoding an HA antigen selected from the group consisting of the following: The mRNA encoding the HA antigen of influenza A virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of influenza B virus. 2. The composition according to claim 1, further comprising a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different strains. 3. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:2 (w / w). 4. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:3 (w / w). 5. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:4 (w / w). 6. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:5 (w / w). The composition described in paragraph 1, wherein mRNA 7.1, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:6 (w / w). 8. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:7 (w / w). 9. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:8 (w / w). 10. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:9 (w / w). 11. The composition described in paragraph 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:10 (w / w). 12. The composition described in paragraph 1, wherein the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w). 13. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 (w / w). 14. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:3:3 (w / w). 15. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:4:4 (w / w). 16. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:5:5 (w / w). 17. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:6:6 (w / w). 18. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:7:7 (w / w). 19. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:8:8 (w / w). 20. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:9:9 (w / w). 21. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:10:10 (w / w). 22. The composition described in paragraph 2, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 to approximately 1:1:10:10 (w / w). 23. The ratio is expressed in micrograms (μg) and is one of the compositions described in any one of paragraphs 1 to 22. 24. The composition described in paragraph 13, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 32 micrograms of the first mRNA to approximately 32 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA. 25. The composition described in paragraph 15, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA. 26. The composition described in paragraph 17, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 96 micrograms of the third mRNA to approximately 96 micrograms of the fourth mRNA. 27. A composition according to any one of paragraphs 13 to 23, comprising a total of 130 micrograms of mRNA. 28. A composition according to any one of paragraphs 13 to 23, comprising a total of 160 micrograms of mRNA. 29. A composition according to any one of paragraphs 13 to 23, comprising a total of 200 micrograms of mRNA. 30. A composition according to any one of paragraphs 13 to 23, comprising a total of 224 micrograms of mRNA. 31. A composition according to any one of paragraphs 13 to 23, comprising a total of 130 micrograms to 224 micrograms of mRNA. 32. The composition according to any one of paragraphs 1 to 31, wherein the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other. 33. The composition according to any one of paragraphs 1 to 31, wherein one or more of the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are covalently bonded to each other. 34. The composition described in any one of paragraphs 1 to 33, wherein the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are incorporated into the LNP. 35. The composition according to paragraph 34, wherein LNP comprises at least one cationic lipid. 36. The composition described in paragraph 35, wherein the cationic lipid is biodegradable. 37. The cationic lipid is not biodegradable, as described in paragraph 35. 38. The composition according to paragraph 35, wherein the cationic lipid is cleavable. 39. The composition described in paragraph 35, wherein the cationic lipid is not cleavable. 40. The composition according to paragraph 35, 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, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), and IM-001. 41. The composition described in paragraph 40, wherein the cationic lipid is cKK-E10. 42. The composition described in paragraph 40, wherein the cationic lipid is GL-HEPES-E3-E12-DS-4-E10. 43. The composition described in paragraph 40, wherein the cationic lipid is IM-001. 44. LNP is a composition according to any one of paragraphs 34 to 43, further comprising polyethylene glycol (PEG) conjugate (PEGylated) lipids, cholesterol-based lipids, and helper lipids. 45. LNP is, Cationic lipids in a molar ratio of 35% to 55%; Polyethylene glycol (PEG) conjugate (PEG-modified) lipids in a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids in a molar ratio of 20% to 45%; and Helper lipids in a molar ratio of 5% to 35% including (Here, all molar ratios are relative to the total lipid content of LNP.) The composition described in any one of paragraphs 34 to 44. 46. ​​LNP is, Cationic lipids at a molar ratio of 40%; PEGylated lipids at a molar ratio of 1.5%; Cholesterol-based lipids in a molar ratio of 28.5%; and Helper lipids at a 30% molar ratio including, The composition described in paragraph 45 (wherein all molar ratios are relative to the total lipid content of LNP). 47. The composition according to any one of paragraphs 44 to 46, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). 48. The cholesterol-based lipid is cholesterol, as described in any one of paragraphs 44 to 47. 49. The composition according to any one of paragraphs 44 to 48, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 50. LNP is, GL-HEPES-E3-E12-DS-4-E10 with a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio including (Hereinafter, all molar ratios are relative to the total lipid content of LNP), the composition described in any one of paragraphs 34 to 49. 51. LNP is, cKK-E10 with a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio including (Hereinafter, all molar ratios are relative to the total lipid content of LNP), the composition described in any one of paragraphs 34 to 49. 52. LNP is, IM-001 with a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio including (Hereinafter, all molar ratios are relative to the total lipid content of LNP), the composition described in any one of paragraphs 34 to 49. 53. The composition according to any one of paragraphs 34 to 52, wherein the LNP has an average diameter of 30 nm to 200 nm. 54. The composition described in paragraph 53, wherein the LNP has an average diameter of 80 nm to 150 nm. 55. The composition described in any one of paragraphs 1 to 54, wherein the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A. 56. The composition described in any one of paragraphs 1 to 55, wherein the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A. 57. A composition according to any one of paragraphs 1 to 56, wherein the third mRNA encodes the HA antigen of a Victoria strain of influenza B. 58. The composition described in any one of paragraphs 2 to 57, wherein the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B. 59. A composition according to any one of paragraphs 1 to 58, wherein at least one mRNA comprises a codon-optimized ORF. 60. A composition according to any one of paragraphs 1 to 59, wherein at least one mRNA 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. 61. A composition according to any one of paragraphs 1 to 60, wherein at least one mRNA comprises at least one chemical modification. 62. The composition according to any one of paragraphs 1 to 61, 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 mRNA are chemically modified. 63. The composition according to any one of paragraphs 1 to 62, 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 ORF are chemically modified. 64. The chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thio-pseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine, as described in any one of paragraphs 50 to 52. 65. The composition described in paragraph 64, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. 66. The composition described in paragraph 65, wherein the chemical modification is N1-methylpseudolidine. 67. A method comprising administering a composition described in any one of paragraphs 1 to 66 to a subject in need thereof. 68. A method for inducing an immune response to influenza A or protecting a subject from influenza A infection, comprising administering to the subject a composition described in any one of paragraphs 1 to 66. 69. A method for inducing an immune response to influenza B or protecting a subject from influenza B infection, comprising administering to a subject one of the compositions described in any one of paragraphs 1 to 66. 70. The method according to paragraph 68, wherein the serum concentration of neutralizing antibodies against influenza A after administration of the composition is equivalent to that of subjects administered with a protein-based influenza A vaccine. 71. The method according to paragraph 69, wherein the serum concentration of neutralizing antibodies against influenza B after administration of the composition is equivalent to that of subjects administered with a protein-type B influenza vaccine. 72. The method according to paragraph 68, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A in a subject. 73. The method according to paragraph 69, wherein the composition increases the serum concentration of neutralizing antibodies against influenza B in a subject. 74. The method according to paragraph 72, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in a subject. 75. The method according to paragraph 73, wherein the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in a subject. 76. A composition according to any one of paragraphs 1 to 66, for use in a method for inducing an immune response to influenza A or for protecting a subject from infection with influenza A. 77. A composition according to any one of paragraphs 1 to 66, for use in a method for inducing an immune response to influenza B or for protecting a subject from infection with influenza B. 78. Use of any one of paragraphs 1 to 66 in the manufacture of a medicinal product for inducing an immune response to influenza A or for protecting a subject from infection with influenza A. 79. Use of any one of paragraphs 1 to 66 in the manufacture of a medicinal product for inducing an immune response to influenza B or for protecting a subject from infection with influenza B. 80. A composition comprising at least three messenger RNAs (mRNAs), (i) The first mRNA encodes the hemagglutinin (HA) antigen of the first influenza A virus; (ii) The second mRNA encodes the HA antigen of the second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) The third mRNA encodes the HA antigen of the first influenza B virus, The mRNA encoding the HA antigen of influenza A virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of influenza B virus, and The first mRNA, the second mRNA, and the third mRNA are, 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 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio A composition incorporated into lipid nanoparticles (LNPs) containing [the specified substance]. 81. Further comprising a fourth mRNA encoding the HA antigen of the second influenza B virus, the first influenza B virus and the second influenza B virus are of different lineages, and the first mRNA, second mRNA, third mRNA, and fourth mRNA are, 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 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio The composition described in paragraph 80, which is incorporated into lipid nanoparticles (LNPs) containing the above. 82. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:2 (w / w). 83. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:3 (w / w). 84. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:4 (w / w). The composition described in paragraph 80, wherein mRNA 85.1, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:5 (w / w). 86. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:6 (w / w). 87. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:7 (w / w). 88. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:8 (w / w). 89. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:9 (w / w). 90. The composition described in paragraph 80, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:10 (w / w). 91. The composition described in paragraph 80, wherein the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w). 92. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 (w / w). 93. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:3:3 (w / w). 94. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:4:4 (w / w). 95. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:5:5 (w / w). 96. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:6:6 (w / w). 97. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:7:7 (w / w). 98. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:8:8 (w / w). 99. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:9:9 (w / w). 100. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:10:10 (w / w). 101. The composition described in paragraph 81, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 to approximately 1:1:10:10 (w / w). 102. The ratio is expressed in micrograms (μg) and is one of the compositions described in any one of paragraphs 80 to 101. 103. The composition described in paragraph 92, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 32 micrograms of the first mRNA to approximately 32 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA. 104. The composition described in paragraph 94, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA. 105. The composition described in paragraph 96, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 16 micrograms of the first mRNA to approximately 16 micrograms of the second mRNA to approximately 96 micrograms of the third mRNA to approximately 96 micrograms of the fourth mRNA. 106. A composition according to any one of paragraphs 92-102, comprising a total of 130 micrograms of mRNA. 107. A composition according to any one of paragraphs 92-102, comprising a total of 160 micrograms of mRNA. 108. A composition according to any one of paragraphs 92-102, comprising a total of 200 micrograms of mRNA. 109. A composition according to any one of paragraphs 92-102, comprising a total of 224 micrograms of mRNA. 110. A composition according to any one of paragraphs 92 to 102, comprising a total of 130 micrograms to 224 micrograms of mRNA. 111. A composition according to any one of paragraphs 80 to 110, wherein at least one mRNA comprises a codon-optimized open reading frame (kORF). 112. A composition according to any one of paragraphs 80 to 111, wherein at least one mRNA 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. 113. A composition according to any one of paragraphs 80 to 112, wherein at least one mRNA comprises at least one chemical modification. 114. The composition according to any one of paragraphs 80 to 113, wherein the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other. 115. The composition according to any one of paragraphs 80 to 113, wherein one or more of the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are covalently bonded to each other. 116. The LNP is a composition according to any one of paragraphs 80 to 115, having an average diameter of 30 nm to 200 nm. 117. The composition described in paragraph 116, wherein the LNP has an average diameter of 80 nm to 150 nm. 118. The composition described in any one of paragraphs 80-117, wherein the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A. 119. The composition described in any one of paragraphs 80-118, wherein the second mRNA encodes the HA antigen of influenza A H3N2 subtype. 120. The composition described in any one of paragraphs 80-119, wherein the third mRNA encodes the HA antigen of the Victoria lineage strain of influenza B. 121. The fourth mRNA is a composition described in any one of paragraphs 81 to 120, encoding the HA antigen of the Yamagata strain of influenza B. 122. A method comprising administering a composition described in any one of paragraphs 80 to 121 to a subject in need thereof. 123. A method for inducing an immune response to influenza A or protecting a subject from influenza A infection, comprising administering to a subject a composition described in any one of paragraphs 80 to 121. 124. A method for inducing an immune response to influenza B or protecting a subject from influenza B infection, comprising administering to a subject a composition described in any one of paragraphs 80 to 121. 125. The method according to paragraph 123, wherein the serum concentration of neutralizing antibodies against influenza A after administration of the composition is equivalent to that of subjects administered with a protein-type A influenza vaccine. 126. The method according to paragraph 124, wherein the serum concentration of neutralizing antibodies against influenza B after administration of the composition is equivalent to that of subjects administered with a protein-type B influenza vaccine. 127. The method according to paragraph 123, wherein the product increases the serum concentration of neutralizing antibodies against influenza A in a subject. 128. The method according to paragraph 124, wherein the composition increases the serum concentration of neutralizing antibodies against influenza B in a subject. 129. The method according to paragraph 123, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in a subject. 130. The method according to paragraph 124, wherein the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in a subject. 131. A composition according to any one of paragraphs 80 to 121, for use in a method for inducing an immune response to influenza A or for protecting a subject from infection with influenza A. 132. A composition according to any one of paragraphs 80 to 121, for use in a method for inducing an immune response to influenza B or for protecting a subject from infection with influenza B. 133. Use of any one of paragraphs 80 to 121 in the manufacture of a medicinal product for inducing an immune response to influenza A or for protecting a subject from infection with influenza A. 134. Use of any one of paragraphs 80 to 121 in the manufacture of a medicinal product for inducing an immune response to influenza B or for protecting a subject from infection with influenza B. 135. A method comprising administering a composition containing at least three messenger RNAs (mRNAs) to a human subject, (i) The first mRNA encodes the hemagglutinin (HA) antigen of the first influenza A virus; (ii) The second mRNA encodes the HA antigen of the second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) The third mRNA encodes the HA antigen of the first influenza B virus, The mRNA encoding the HA antigen of influenza A virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of influenza B virus, and the first mRNA, second mRNA, and third mRNA are... 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 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio A method for incorporating lipid nanoparticles (LNPs) containing [the specified substance]. 136. The composition comprises administering a composition comprising a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different lineages, and the first mRNA, second mRNA, third mRNA, and fourth mRNA are, 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 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE with a 30% molar ratio The method described in paragraph 135, which is incorporated into lipid nanoparticles (LNPs) containing [the specified substance]. 137. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:2 (w / w). 138. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:3 (w / w). 139. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:4 (w / w). 140. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:5 (w / w). 141. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:6 (w / w). 142. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:7 (w / w). 143. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:8 (w / w). 144. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:9 (w / w). 145. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:10 (w / w). 146. The composition of paragraph 135, in which the first mRNA, second mRNA, and third mRNA are present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w). 147. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 (w / w). 148. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:3:3 (w / w). 149. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:4:4 (w / w). 150. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:5:5 (w / w). The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:6:6 (w / w). 152. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:7:7 (w / w). 153. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:8:8 (w / w). The composition of paragraph 136, in which mRNA 154.1, the second mRNA, the third mRNA, and the fourth mRNA are present in a ratio of approximately 1:1:9:9 (w / w). 155. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:10:10 (w / w). 156. The composition of paragraph 136, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 to approximately 1:1:10:10 (w / w). 157. The ratio is expressed in micrograms (μg) as described in any one of paragraphs 135-156. 158. The first, second, third, and fourth mRNAs are present in the ratio (w / w) of approximately 32 micrograms of the first mRNA to approximately 32 micrograms of the second mRNA to approximately 64 micrograms of the third mRNA to approximately 64 micrograms of the fourth mRNA, according to the method of paragraph 147. 159. The first, second, third, and fourth mRNAs are present in the ratio (w / w) of approximately 16 micrograms of the first mRNA, 16 micrograms of the second mRNA, 64 micrograms of the third mRNA, and 64 micrograms of the fourth mRNA, according to the method of paragraph 149. 160. The first, second, third, and fourth mRNAs are present in the ratio (w / w) of approximately 16 micrograms of the first mRNA, 16 micrograms of the second mRNA, 96 micrograms of the third mRNA, and 96 micrograms of the fourth mRNA, according to the method of paragraph 151. 161. The method according to any one of paragraphs 147-157, wherein the composition contains a total of 130 micrograms of mRNA. 162. The method according to any one of paragraphs 147-157, wherein the composition contains a total of 160 micrograms of mRNA. 163. The method according to any one of paragraphs 147-157, wherein the composition contains a total of 200 micrograms of mRNA. 164. The method according to any one of paragraphs 147-157, wherein the composition contains a total of 224 micrograms of mRNA. 165. The method according to any one of paragraphs 147 to 157, wherein the composition contains a total of 130 to 224 micrograms of mRNA. 166. The method described in any one of paragraphs 135–165, wherein at least one mRNA contains a codon-optimized open reading frame (ORF). 167. The method according to any one of paragraphs 135-166, wherein at least one mRNA 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. 168. The method described in any one of paragraphs 135-167, wherein at least one mRNA contains at least one chemical modification. 169. The method described in any one of paragraphs 135-168, wherein the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other. 170. The method according to any one of paragraphs 135-168, wherein one or more of the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are covalently bonded to each other. 171. An LNP having an average diameter of 30 nm to 200 nm, according to any one of the methods described in paragraphs 135 to 170. 172. The LNP having an average diameter of 80 nm to 150 nm, as described in paragraph 171. 173. The first mRNA encodes the HA antigen of influenza A H1N1 subtype, as described in any one of paragraphs 135-172. 174. The second mRNA encodes the HA antigen of influenza A H3N2 subtype, as described in any one of paragraphs 135-173. 175. The third mRNA encodes the HA antigen of the influenza B Victoria lineage strain, as described in any one of paragraphs 135-174. 176. The fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B, as described in any one of paragraphs 136-175. 177. The method according to any one of paragraphs 135 to 176, wherein the composition is administered in an amount effective to induce an immune response to influenza A or to protect the subject from influenza A infection. 178. The method according to any one of paragraphs 135-176, wherein the product is administered in an amount effective to induce an immune response to influenza B or to protect the subject from influenza B infection. 179. The method according to any one of paragraphs 135 to 176, wherein the subject has a serum concentration of neutralizing antibodies against influenza A after administration of the composition, compared to a subject administered with a protein-type A influenza vaccine. 180. The method according to any one of paragraphs 135 to 176, wherein the subjects have serum concentrations of neutralizing antibodies against influenza B after administration of the composition, compared to subjects who have been administered a protein-type B influenza vaccine. 181. The method according to any one of paragraphs 135 to 176, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A in a subject. 182. A composition according to any one of paragraphs 135 to 176, which increases the serum concentration of neutralizing antibodies against influenza B in a subject. 183. The method according to paragraph 181, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in a subject. 184. The method according to paragraph 182, wherein the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in a subject. 185. A composition comprising at least three messenger RNAs (mRNAs), (i) The first mRNA encodes the hemagglutinin (HA) antigen of the first influenza A virus; (ii) The second mRNA encodes the HA antigen of the second influenza A virus (where the first influenza A virus and the second influenza A virus are different subtypes); and (iii) The third mRNA encodes the HA antigen of the first influenza B virus, The first mRNA, the second mRNA, and the third mRNA are compositions incorporated into lipid nanoparticles (LNPs) containing IM-001. 186. The composition according to paragraph 185, further comprising a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different lineages, and the first mRNA, second mRNA, third mRNA, and fourth mRNA are incorporated into an LNP containing IM-001. 187. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:2 (w / w). 188. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:3 (w / w). 189. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:4 (w / w). 190. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:5 (w / w). 191. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:6 (w / w). 192. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:7 (w / w). 193. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:8 (w / w). 194. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:9 (w / w). 195. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:10 (w / w). 196. The composition described in paragraph 185, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio of approximately 1:1:2 to approximately 1:1:10 (w / w). 197. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 (w / w). 198. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:3:3 (w / w). 199. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:4:4 (w / w). 200. The first mRNA, second mRNA, third mRNA, and fourth mRNA are in a ratio of approximately 1:1:5:5. The composition of paragraph 186, which exists in w / w form. 201. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:6:6 (w / w). 202. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:7:7 (w / w). 203. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:8:8 (w / w). 204. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:9:9 (w / w). 205. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:10:10 (w / w). 206. The composition of paragraph 186, in which the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio of approximately 1:1:2:2 to approximately 1:1:10:10 (w / w). 207. The ratio is expressed in micrograms (μg) and is one of the compositions described in any one of paragraphs 187 to 205. 208. The composition of paragraph 197, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 32 micrograms of first mRNA to approximately 32 micrograms of second mRNA to approximately 64 micrograms of third mRNA to approximately 64 micrograms of fourth mRNA. 209. The composition of paragraph 199, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 16 micrograms of first mRNA to approximately 16 micrograms of second mRNA to approximately 64 micrograms of third mRNA to approximately 64 micrograms of fourth mRNA. 210. The composition of paragraph 201, wherein the first mRNA, second mRNA, third mRNA, and fourth mRNA are present in a ratio (w / w) of approximately 16 micrograms of the first mRNA, approximately 16 micrograms of the second mRNA, approximately 96 micrograms of the third mRNA, and approximately 96 micrograms of the fourth mRNA. 211. A composition according to any one of paragraphs 187-207, comprising a total of 130 micrograms of mRNA. 212. A composition according to any one of paragraphs 187-207, comprising a total of 160 micrograms of mRNA. 213. A composition according to any one of paragraphs 187-207, comprising a total of 200 micrograms of mRNA. 214. A composition according to any one of paragraphs 187-207, comprising a total of 224 micrograms of mRNA. 215. A composition according to any one of paragraphs 187 to 207, comprising a total of 130 micrograms to 224 micrograms of mRNA. 216. The composition according to any one of paragraphs 1 to 215, wherein the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are not covalently bonded to each other. 217. The composition according to any one of paragraphs 185 to 215, wherein one or more of the first mRNA, second mRNA, third mRNA, and / or fourth mRNA are covalently bonded to each other. 218. LNP is a composition according to any one of paragraphs 1 to 217, further comprising polyethylene glycol (PEG) conjugate (PEGylated) lipids, cholesterol-based lipids, and helper lipids. 219LNP is, IM-001 with a molar ratio of 35% to 55%; Polyethylene glycol (PEG) conjugate (PEG-modified) lipids in a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids in a molar ratio of 20% to 45%; and Helper lipids in a molar ratio of 5% to 35% including (Here, all molar ratios are relative to the total lipid content of LNP.) A composition described in any one of paragraphs 1 to 218. 220.LNP is, IM-001 with a molar ratio of 40%; PEGylated lipids at a molar ratio of 1.5%; Cholesterol-based lipids in a molar ratio of 28.5%; and Helper lipids at a 30% molar ratio including (Here, all molar ratios are relative to the total lipid content of LNP.) The composition described in paragraph 219. 221. The composition according to any one of paragraphs 218 to 220, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). 222. The cholesterol-based lipid is cholesterol, as described in any one of paragraphs 218 to 221. 223. The composition according to any one of paragraphs 218 to 222, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 224. The LNP is a composition according to any one of paragraphs 1 to 223, having an average diameter of 30 nm to 200 nm. 225. The composition described in paragraph 224, wherein the LNP has an average diameter of 80 nm to 150 nm. 226. The composition described in any one of paragraphs 185 to 225, wherein the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A. 227. The composition described in any one of paragraphs 185 to 226, wherein the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A. 228. The composition described in any one of paragraphs 185-227, wherein the third mRNA encodes the HA antigen of the Victoria strain of influenza B. 229. The composition described in any one of paragraphs 186 to 229, wherein the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B. 230. A composition according to any one of paragraphs 185-229, wherein at least one mRNA comprises a codon-optimized ORF. 231. A composition according to any one of paragraphs 185 to 230, wherein at least one RNA 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. 232. A composition according to any one of paragraphs 185 to 231, wherein at least one mRNA comprises at least one chemical modification. 233. A composition according to any one of paragraphs 185 to 232, 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 mRNA are chemically modified. 234. The composition according to any one of paragraphs 185 to 233, 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 ORF are chemically modified. 235. The chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thio-pseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine, as described in any one of paragraphs 232 to 234. 236. The composition described in paragraph 235, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. 237. The composition described in paragraph 236, wherein the chemical modification is N1-methylpseudolidine. 238. A method comprising administering a composition described in any one of paragraphs 185 to 237 to a subject in need thereof. 239. A method for inducing an immune response to influenza A or protecting a subject from influenza A infection, comprising administering to a subject a composition described in any one of paragraphs 185 to 237. 240. A method for inducing an immune response to influenza B or protecting a subject from influenza B infection, comprising administering to a subject a composition described in any one of paragraphs 185 to 237. 241. The method according to paragraph 239, wherein the serum concentration of neutralizing antibodies against influenza A after administration of the composition is equivalent to that of subjects administered with a protein-based influenza A vaccine. 242. The method according to paragraph 240, wherein the serum concentration of neutralizing antibodies against influenza B after administration of the composition is equivalent to that of subjects administered with a protein-type B influenza vaccine. 243. The method according to paragraph 239, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A in a subject. 244. The method according to paragraph 240, wherein the composition increases the serum concentration of neutralizing antibodies against influenza B in a subject. 245. The method according to paragraph 243, wherein the composition increases the serum concentration of neutralizing antibodies against influenza A H1N1 subtype and / or influenza A H3N2 subtype in a subject. 246. The method according to paragraph 244, wherein the composition increases the serum concentration of neutralizing antibodies against the Yamagata and / or Victoria lineages of influenza B in a subject. 247. A composition according to any one of paragraphs 185 to 237, for use in a method of inducing an immune response to influenza A or in a method of protecting a subject from infection with influenza A. 248. A composition according to any one of paragraphs 185 to 237, for use in a method of inducing an immune response to influenza B or in a method of protecting a subject from infection with influenza B. 249. Use of any one of the compositions described in paragraphs 185 to 237 in the manufacture of a medicinal product for inducing an immune response to influenza A or for protecting a subject from infection with influenza A. 250. Use of any one of the compositions described in paragraphs 185 to 237 in the manufacture of a medicinal product for inducing an immune response to influenza B or for protecting a subject from infection with influenza B. [Examples]

[0654] Example 1: 1:1:1:1 Combination influenza mRNA vaccine formulation: Immunogenicity in mice This example describes experiments evaluating the efficacy of monovalent and quadrivalent mRNA-LNP vaccine formulations containing mRNA encoding the HA sequences of two influenza A strains (i.e., A / H1N1, A / H3N2) and two influenza B strains (i.e., B / Yamagata lineage and B / Victoria lineage) in a 1:1:1:1 ratio. Immunogenicity of three quadrivalent modified mRNA influenza vaccines encapsulated in LNP formulations containing cKK-E10 at a 40% molar ratio; DMG-PEG2000 at a 1.5% molar ratio; cholesterol at a 28.5% molar ratio; and DOPE at a 30% molar ratio. 40 micrograms of mRNA encoding HA-1 (A / Michigan / 45 / 2015), HA-3 (A / Singapore / INFIMH160019 / 2016), HA-B Victoria (B / Maryland / 15 / 2016BX69A), and HA-B Yamagata (B / Phuket / 3073 / 2013) were administered to BALB / c mice twice, 3 weeks apart, either individually or together (40 micrograms of each mRNA preparation were mixed before administration). Samples were collected on days 1, 20, 22, and 36. Hemagglutination inhibition (HAI) response to homologous strains was evaluated to measure functional antibodies. The results for the day 36 sample are shown in Table 1 below.

[0655] [Table 5]

[0656] No significant difference was observed between the monovalent and quadrivalent vaccines in terms of immune response to any of the four antigens. However, all HAI GMTs from strain B were at the detection limit, indicating a lower immune response compared to strain A.

[0657] Example 2: 1:1:1:1 combination influenza mRNA vaccine formulation in human clinical trials Pandemic influenza can occur when a novel influenza virus emerges in a human population. Such pandemics still pose a significant threat to public health and require vigilance and preparedness with measures to be used if human-to-human transmission of the virus persists. In the experiments described in this embodiment, three Phase I / II trials were designed to evaluate the safety and immunogenicity of three quadrivalent modified mRNA influenza vaccines encapsulated in LNP formulations containing the HA sequences of two influenza A strains (i.e., A / H1N1 and A / H3N2) and two influenza B strains (i.e., B / Yamagata lineage and B / Victoria lineage) in a 1:1:1:1:1 ratio, and one of three cationic lipids: GL-HEPES-E3-E12-DS-4-E10 (clinical trial NCT05624606), ckK-E10 (clinical trial NCT05553301), or OF-02 (clinical trial NCT05650554). The LNP was designed as follows: 40% molar ratio of cationic lipid; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE.

[0658] Clinical trial NCT05624606 evaluated the safety and immunogenicity of the quadrivalent influenza mRNA vaccine MRT5410 in adults aged 18 years and older, administered as a single intramuscular injection at three dose levels (i.e., low, medium, and high), compared to the following active controls: (1) Fluzone Quadrivalent®, standard dose quadrivalent inactivated influenza vaccine, and comparison subjects in trial participants aged 18–64 years and 65 years and older; (2) Flublok Quadrivalent®, quadrivalent recombinant influenza vaccine, and comparison subjects in trial participants aged 18–64 years and 65 years and older; and (3) Fluzone High-Dose Quadrivalent®, high dose quadrivalent inactivated influenza vaccine, and comparison subjects in trial participants aged 65 years and older only.

[0659] Clinical trial NCT05553301 will evaluate the safety and immunogenicity of the quadrivalent influenza mRNA vaccine MRT5407 in adults aged 18 years and older, administered as a single intramuscular injection at two dose levels (i.e., dose level 1 and dose level 2), compared to the following active controls: (1) Fluzone Quadrivalent®, a comparison group for participants aged 18–64 years and 65 years and older; (2) Flublok Quadrivalent®, a comparison group for participants aged 18–64 years and 65 years and older; and (3) Fluzone High-Dose Quadrivalent®, a comparison group for participants aged 65 years and older only.

[0660] Clinical trial NCT05650554 evaluated the safety and immunogenicity of the quadrivalent influenza mRNA vaccine MRT5413 in adults aged 18 years and older, administered as a single intramuscular injection at three dose levels (i.e., low, medium, and high), compared to the following active controls: (1) Fluzone Quadrivalent®, a comparison group for participants aged 18–64 years and 65 years and older; (2) Flublok Quadrivalent®, a comparison group for participants aged 18–64 years and 65 years and older; and (3) Fluzone High-Dose Quadrivalent®, a comparison group for participants aged 65 years and older only.

[0661] Interim results from clinical trial NCT05624606 show that administration of a 1:1:1:1 quadrivalent mRNA vaccine formulated with the cationic lipid GL-HEPES-E3-E12-DS-4-E10 generated hemagglutinin inhibitory (HAI) antibodies against all four influenza strains at all three dose levels (i.e., low, medium, and high) in adults aged 18–64 years. Furthermore, as shown in Figure 1, the immune response to influenza A strains (e.g., A / H1N1, A / H3N2) was comparable to that of the control group in adults aged 18–64 years, but a lower immune response was observed against influenza B strains (e.g., B / Yamagata lineage and B / Victoria lineage).

[0662] Interim results from clinical trial NCT05553301 show that in adults aged 18–64 years, administration of a 1:1:1:1 quadrivalent mRNA vaccine formulated with the cationic lipid ckk-E10 generated HAI antibodies against all four influenza strains at both dose levels (i.e., dose level 1 and dose level 2). Furthermore, as shown in Figure 2, in adults aged 18–64 years, the immune response to influenza A strains (e.g., A / H1N1, A / H3N2) was comparable to that of the control group, but a lower immune response was observed against influenza B strains (e.g., B / Yamagata lineage and B / Victoria lineage).

[0663] The data from the above trials indicate that there is a need to improve the immunogenicity against strain B in combination influenza mRNA vaccine formulations.

[0664] Example 3: Improvement of B strain immunogenicity in combination influenza mRNA vaccine formulations One exemplary strategy for improving the immunogenicity of influenza B strains in combination influenza mRNA vaccine formulations is to increase the ratio / amount of mRNA encoding the HA sequence of influenza B virus compared to mRNA encoding the HA sequence of influenza A virus.

[0665] Table 2 below shows an example of a clinical trial protocol in which eligible participants (adults aged 18 to 64 years and 65 years or older) are randomly assigned to receive one of the following single intramuscular injections: (1) a 1:1:1:1 quadrivalent mRNA vaccine encoding the HA sequences of influenza A and B strains, total mRNA dose 130 μg; (2) a 1:1:4:4 quadrivalent mRNA vaccine encoding the HA sequences of influenza A and B strains, total mRNA dose 160 μg; (3) a 1:1:2:2 quadrivalent mRNA vaccine encoding the HA sequences of influenza A and B strains, total mRNA dose 200 μg; (4) a 1:1:6:6 quadrivalent mRNA vaccine encoding the HA sequences of influenza A and B strains, total mRNA dose 224 μg; (5) one dose of Fluzone Quadrivalent® influenza vaccine (standard dose) (Sanofi Pasteur); (6) one dose of Fluzone High-Dose Quadrivalent® influenza vaccine (Sanofi Pasteur); or (7) one dose of Flublok Quadrivalent® recombinant influenza vaccine (Sanofi Pasteur).

[0666] [Table 6]

[0667] The purpose of this study is to evaluate the safety and immunogenicity of quadrivalent influenza mRNA vaccines with different A:A:B:B HA mRNA ratios and total mRNA doses, administered as a single intramuscular injection to adults aged 18 years or older, compared to the following controls: one dose of Fluzone Quadrivalent® influenza vaccine (standard dose) to participants aged 18–64 years and 65 years or older; one dose of Fluzone High-Dose Quadrivalent® influenza vaccine to participants aged 65 years or older only; and one dose of Flublok Quadrivalent® recombinant influenza vaccine to participants aged 18–64 years and 65 years or older.

[0668] Table 3 below shows alternative clinical trial protocols for randomly assigning eligible participants (adults aged 18 to 64 years and 65 years or older) to receive one of the following single intramuscular injections: (1) a 1:1:2:2 quadrivalent mRNA vaccine encoding HA sequences of influenza A and B strains, total mRNA dose of 192 μg, in an LNP containing the cationic lipid GL-HEPES-E3-E12-DS-4-E10; (2) a 1:1:2:2 quadrivalent mRNA vaccine encoding HA sequences of influenza A and B strains, total mRNA dose of 30 μg, in an LNP containing the cationic lipid IM-01; (3) a 1:1:2:2 quadrivalent mRNA vaccine encoding HA sequences of influenza A and B strains, total mRNA dose of 96 μg, in an LNP containing the cationic lipid IM-01; (4) influenza A (5) A 1:1:2:2 quadrivalent mRNA vaccine encoding the HA sequences of influenza strains and influenza B strains, with a total mRNA dose of 132 μg, contained in an LNP with cationic lipid IM-01; (6) A 1:1:2:2 quadrivalent mRNA vaccine encoding the HA sequences of influenza A strains and influenza B strains, with a total mRNA dose of 192 μg, contained in an LNP with cationic lipid IM-01; (7) A 1:1:2:2 quadrivalent mRNA vaccine encoding the HA sequences of influenza A strains and influenza B strains, with a total mRNA dose of 96 μg, contained in an LNP with cationic lipid ALC-0315; (5) One dose of Fluzone (6) One dose of Quadrivalent® influenza vaccine (standard dose) QIV-SD (Sanofi Pasteur); or (7) One dose of Flublok Quadrivalent® recombinant influenza vaccine RIV (Sanofi Pasteur).

[0669] [Table 7]

[0670] The purpose of this study is to evaluate the safety and immunogenicity of quadrivalent influenza mRNA vaccines with different A:A:B:B HA mRNA ratios and total mRNA doses among various LNP formulations administered as a single intramuscular injection to adults aged 18 years or older, compared to the following controls: one dose of Fluzone Quadrivalent® influenza vaccine (standard dose) as a comparison group for participants aged 18–64 years and 65 years or older; one dose of Fluzone High-Dose Quadrivalent® influenza vaccine as a comparison group for participants aged 65 years or older only; and one dose of Flublok Quadrivalent® recombinant influenza vaccine as a comparison group for participants aged 18–64 years and 65 years or older.

[0671] The following are exemplary nucleic acid sequences encoding exemplary influenza A and influenza B constructs that can be used in clinical trial protocols.

[0672] A / Wisconsin / 588 / 2019 [ka] [ka]

[0673] A / Tasmania / 503 / 2020 [ka] [ka]

[0674] B / Washington / 02 / 2019 [ka] [ka]

[0675] B / Phuket / 3073 / 2013 [ka] [ka]

[0676] Example 4: Use of IM-001 containing LNP to induce an HA immune response LNPs containing 40% molar ratio IM-001; 1.5% molar ratio DMG-PEG2000; 28.5% molar ratio cholesterol; and 30% molar ratio DOPE were used to deliver the HA antigen to mice. Balb / c mice (Mus musculus) were immunized under isoflurane anesthesia in each treatment group by administering a dose of 0.4 μg per mouse in 0.05 mL of IM-001 / modified Tasmania H3 mRNA-lipid nanoparticles via the IM pathway in the quadriceps femoris muscle, to one hind leg on day 0 and to the opposite hind leg on day 21. Mice were evaluated for at least 3 days after administration, and animals showing severe clinical symptoms after veterinary evaluation were euthanized by subcutaneous injection of meloxicam 5 mg / kg.

[0677] Blood was collected from all sedated animals via submandibular or orbital sinus hemorrhage (in vivo hemorrhage was performed on days -1 and 20) and cardiac puncture (peripheral hemorrhage, day 35). Blood was collected from mice before the study to obtain baseline preimmune serum samples and for pre-screening purposes.

[0678] HAI assays were performed using the A / Tasmania / 503 / 2020(H3N2) virus stock (BIOQUAL, Inc.). Serum was treated with receptor-destroying enzyme (RDE), and a portion of the serum was diluted with 3 parts of the enzyme and incubated overnight in a 37°C water bath. The enzyme was inactivated by incubation at 56°C for 30 minutes, followed by the addition of 6 parts of PBS to obtain 1 / 10 of the final dilution. HAI assays were performed in V-bottom 96-well plates using four hemagglutination units (HAU) of the virus and 0.5% turkey RBCs. Reference serum for each strain was included in each assay plate as a positive control. Each plate also included back titration to confirm the antigen dose (4 HAU / 25 pl) and negative control sample (PBS or naive control serum). HAI titer was determined as the highest serum dilution at which hemagglutination was completely inhibited. The results were effective only on plates with appropriate back titration results (verifying the addition of 4 HAU / 25 μl) and reference serum titers within twice the expected titer. The vaccine was shown to induce 320 HAI GMTs, demonstrating the induction of high levels of immunogenicity against influenza antigens by these LNPs.

Claims

1. A composition comprising at least three messenger RNAs (mRNAs), wherein the at least three mRNAs are (i) The first mRNA encoding the HA antigen of the first influenza A virus; (ii) a second mRNA encoding the HA antigen of a second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes); and (iii) The third mRNA encoding the HA antigen of the first influenza B virus. It includes an open reading frame (ORF) encoding a hemagglutinin (HA) antigen selected from the group consisting of the following: A composition wherein the mRNA encoding the HA antigen of the influenza A virus is present in a different ratio (w / w) than the mRNA encoding the HA antigen of the influenza B virus.

2. The composition according to claim 1, further comprising a fourth mRNA encoding the HA antigen of a second influenza B virus, wherein the first influenza B virus and the second influenza B virus are of different strains.

3. The composition according to claim 1, wherein the first mRNA, the second mRNA, and the third mRNA are present in a ratio (w / w) of about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9, or about 1:1:

10.

4. The composition according to claim 2, wherein the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA are present in a ratio (w / w) of about 1:1:2:2, about 1:1:3:3, about 1:1:4:4, about 1:1:5:5, about 1:1:6:6, about 1:1:7:7, about 1:1:8:8, about 1:1:9:9, or about 1:1:10:

10.

5. The composition according to any one of claims 1 to 22, wherein the ratio is expressed in micrograms (μg).

6. The composition according to any one of claims 1 to 5, wherein the first mRNA, the second mRNA, the third mRNA, and / or the fourth mRNA are incorporated into the LNP.

7. The composition according to claim 6, wherein the LNP optionally comprises at least one cationic lipid 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, (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), and IM-001.

8. The composition according to claim 6 or 7, wherein the LNP further comprises polyethylene glycol (PEG) conjugate (PEG-modified) lipids, cholesterol-based lipids, and helper lipids.

9. The aforementioned LNP is, Cationic lipids in a molar ratio of 35% to 55%; Polyethylene glycol (PEG) conjugated lipids in a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids in a molar ratio of 20% to 45%; and Helper lipids in a molar ratio of 5% to 35% Includes (Here, all of the above molar ratios are relative to the total lipid content of the LNP.) By choice, the LNP is, Cationic lipids at a molar ratio of 40%; PEG-treated lipids at a molar ratio of 1.5%; Cholesterol-based lipids at a molar ratio of 28.5%; and Helper lipids at a 30% molar ratio including (Hereinafter, all molar ratios are in relation to the total lipid content of the LNP), the composition according to any one of claims 6 to 8.

10. The composition according to claim 8 or 9, wherein the PEG-modified lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and / or the cholesterol-based lipid is cholesterol; and / or the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

11. The composition according to any one of claims 1 to 10, wherein the first mRNA encodes the HA antigen of the H1N1 subtype of influenza A, and / or the second mRNA encodes the HA antigen of the H3N2 subtype of influenza A, and / or the third mRNA encodes the HA antigen of the Victoria lineage strain of influenza B.

12. The composition according to any one of claims 2 to 11, wherein the fourth mRNA encodes the HA antigen of the Yamagata strain of influenza B.

13. A composition comprising at least three messenger RNAs (mRNAs), (i) The first mRNA encodes the hemagglutinin (HA) antigen of the first influenza A virus; (ii) The second mRNA encodes the HA antigen of the second influenza A virus (wherein the first influenza A virus and the second influenza A virus are different subtypes); and (iii) The third mRNA encodes the HA antigen of the first influenza B virus, The first mRNA, the second mRNA, and the third mRNA are compositions incorporated into lipid nanoparticles (LNPs) containing IM-001.

14. A composition according to any one of claims 1 to 14, for use in a method of inducing an immune response to influenza A or in a method of protecting a subject from infection with influenza A.

15. A composition according to any one of claims 1 to 14, for use in a method of inducing an immune response to influenza B or in a method of protecting a subject from infection with influenza B.

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