Lipid nanoparticles for delivering mRNA vaccines

Abstract

The present invention provides a composition comprising novel lipid nanoparticles for delivering nucleic acids such as mRNA. [Solution] A pharmaceutical composition comprising nucleic acid molecules encapsulated in lipid nanoparticles (LNPs), wherein the LNPs comprise cationic lipids, polyethylene glycol (PEG) conjugated (PEG-modified) lipids, cholesterol-based lipids, and helper lipids.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 110,965 filed on 6 November 2020, U.S. Provisional Patent Application No. 63 / 212,523 filed on 18 June 2021, and European Priority Patent Application No. 21315198.8 filed on 13 October 2021, with the contents of each of the aforementioned patent documents incorporated in their entirety by reference for all purposes. [Background technology]

[0002] Messenger RNA (mRNA)-based vaccines offer a promising alternative to traditional subunit vaccines containing pathogen-derived antigen proteins. Antigen proteins are typically synthesized recombinantly, requiring bacterial fermentation and / or cell culture, as well as complex purification. mRNA-based vaccines enable the novel expression of complex antigens in the vaccinated target, which in turn allows for appropriate post-translational modification and presentation of the antigen in its native form. Unlike traditional techniques, the production of mRNA vaccines does not require complex and costly bacterial fermentation, tissue culture, and purification methods. Furthermore, once established, the manufacturing methods for mRNA vaccines can be used for a variety of antigens, enabling the rapid development and distribution of mRNA vaccines. In addition, mRNA vaccines are inherently safe delivery vectors because they only transiently express antigens and are not integrated into the host genome. Because the antigen encoded by mRNA is produced in vivo in the vaccinated individual, mRNA vaccines are particularly effective in inducing both humoral and T-cell-mediated immunity. [Overview of the project] [Problems that the invention aims to solve]

[0003] However, RNA is unstable and susceptible to rapid degradation. There are also no natural cell surface receptors to facilitate RNA uptake into cells. In fact, mRNA development has been hampered by the inefficient uptake of mRNA. This has been hindered by vivo delivery. Therefore, there remains a need to develop vaccine formulations that can improve mRNA delivery in vivo. [Means for solving the problem]

[0004] This disclosure provides a pharmaceutical composition comprising nucleic acid molecules (e.g., mRNA molecules) encapsulated in lipid nanoparticles (LNPs), wherein each LNP contains cationic lipids in a molar ratio of 35% to 45%, 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 35%, and helper lipids in a molar ratio of 25% to 35%, all molar ratios being relative to the total lipid content of the LNP. The composition can be used as a vaccine to induce immunodefence in subjects requiring immunodefence (e.g., human subjects).

[0005] In some embodiments, the cationic lipid is 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.

[0006] In some embodiments, the LNP contains cationic lipids in a molar ratio of 40%, pegylated lipids in a molar ratio of 1.5%, cholesterol-based lipids in a molar ratio of 28.5%, and helper lipids in a molar ratio of 30%.

[0007] In some embodiments, the cationic lipid is 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, the pegylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000), the cholesterol-based lipid is cholesterol, and / or the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE). In certain embodiments, the LNP comprises 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 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%.

[0008] In some embodiments, the LNP comprises 1 to 20, and optionally 5 to 10 or 6 to 8, nucleic acid molecules. In some embodiments, the LNP comprises one or more mRNA molecules encoding an antigen (e.g., a viral antigen such as influenza virus antigen, or a bacterial antigen).

[0009] In some embodiments, the LNP comprises two or more mRNA molecules, each mRNA molecule encoding a different antigen, and the different antigens may be from the same pathogen or from different pathogens.

[0010] For example, the composition may contain (i) different hemagglutinin (HA) antigens, (ii) different neuraminidase (NA) antigens, or (iii) two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding at least one HA antigen and at least one NA antigen.

[0011] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) encoding a respiratory syncytial virus (RSV) F protein antigen.

[0012] In some embodiments, the RSV F protein antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 16 or consists of the amino acid sequence of SEQ ID NO: 16.

[0013] In some embodiments, the RSV F protein antigen is a prefusion protein.

[0014] In some embodiments, the ORF is codon optimized.

[0015] In some embodiments, the mRNA molecule comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and at least one polyadenylation (poly(A)) sequence.

[0016] In some embodiments, the mRNA comprises at least one chemical modification.

[0017] 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.

[0018] 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.

[0019] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0020] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0021] In some embodiments, the chemical modification is N1-methylpseudouridine.

[0022] In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence set forth in SEQ ID NO: 17.

[0023] In some embodiments, the mRNA comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence set forth in SEQ ID NO: 21.

[0024] In some embodiments, the mRNA comprises the following structural elements: (i) a 5' cap having the following structure:

Chemical formula

[0025] In some embodiments, the LNPs have an average diameter of 30 to 200 nm (e.g., 80 to 150 nm). In some embodiments, the composition contains 1 to 10, optionally 1 mg / mL, of LNPs. The composition may be formulated for intramuscular or intradermal injection and may contain phosphate-buffered saline. In some embodiments, the composition may optionally contain trehalose at 10% (w / v) of the composition.

[0026] In another aspect, the disclosure provides an aqueous buffer solution containing nucleic acid molecules and cationic This specification provides a method for producing an LNP composition, comprising providing an amphiphilic solution containing lipids, pegylated lipids, cholesterol-based lipids, and helper lipids, and mixing an aqueous buffer solution with the amphiphilic solution in a ratio of 5:1 to 3:1, and optionally 4:1. The aqueous buffer solution may be, for example, an acidic buffer solution (e.g., containing 1 mM citric acid and 150 mM sodium chloride at pH approximately 4.5). The amphiphilic solution may be, for example, an ethanol solution.

[0027] In another embodiment, the Disclosure provides a method for inducing an immune response in a subject requiring such induction, comprising administering a prophylactically effective amount of the LNP composition to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally. In some embodiments, the subject is treated with one or more (e.g., two) doses of the composition, each dose containing 1 to 250, optionally 2.5, 5, 15, 45, or 135 μg of mRNA. The doses may be administered at intervals of 2 to 24, optionally 4, 8, 12, 16, or 20 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0028] Uses of this composition for the manufacture of drugs to be used to treat subjects requiring treatment, as well as compositions to be used to treat subjects requiring treatment, are also provided herein.

[0029] The disclosure also provides kits comprising containers containing single-use or multi-use doses of the composition, the containers being vials or pre-filled syringes or injectors, as may be the case.

[0030] In another aspect, this disclosure describes lipid nanoparticles (LNPs) as: Cationic lipids in a molar ratio of 35% to 45%, Polyethylene glycol (PEG) conjugate lipids in a molar ratio of 0.25% to 2.75% Cholesterol-based lipids in a molar ratio of 20% to 35%, and It contains helper lipids in a molar ratio of 25% to 35%. All molar ratios are relative to the total lipid content of LNP; The present invention provides a pharmaceutical composition comprising an mRNA molecule encapsulated in an LNP, the mRNA molecule containing an open reading frame (ORF) encoding an antigen derived from the influenza virus.

[0031] In another aspect, this disclosure describes lipid nanoparticles (LNPs) as: Cationic lipids in a molar ratio of 35% to 45%, Polyethylene glycol (PEG) conjugate lipids in a molar ratio of 0.25% to 2.75% Cholesterol-based lipids in a molar ratio of 20% to 35%, and It contains helper lipids in a molar ratio of 25% to 35%. All molar ratios are relative to the total lipid content of LNP; The present invention provides a pharmaceutical composition comprising an mRNA molecule encapsulated in an LNP, the mRNA molecule containing an open reading frame (ORF) encoding the respiratory multinuclear virus (RSV) F protein antigen.

[0032] Other functions, purposes, and advantages of the present invention will become apparent in the detailed description that follows. However, it should be understood that the detailed description illustrates, but is not limiting, embodiments and aspects of the present invention, and is given only as examples. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawing]

[0033] [Figure 1A] Figure 1A is a pair of graphs showing the expression of human erythropoietin (hEPO) mRNA in mice treated with various LNP preparations. Panel a): LNP preparations "Lipid A" and "Lipid B" compared to MC3. Bars represent the mean and standard deviation. Panel b): Preparations made with cationic lipid OF-02. PEG: DMG-PEG2000. Cholest: Cholesterol. "Lipid A": Unless otherwise indicated, an LNP composition containing OF-02, DMG-PEG2000, cholesterol, and DOPE in this order in a molar ratio of 40:1.5:28.5:30. "Lipid B": An LNP composition containing cKK-E10, DMG-PEG2000, cholesterol, and DOPE in this order in a molar ratio of 40:1.5:28.5:30. [Figure 1B] Figure 1B is a pair of graphs showing hEPO expression in mice and non-human primates (NHPs) using LNP preparation lipids A and B. [Figure 2A]Figures 2A and 2B are a pair of graphs showing that lipid A and lipid B LNP preparations containing hemagglutinin (HA)-encoding mRNA of strain A / California / 7 / 2009(H1N1)(CA09) induced robust functional antibodies (Figure 2A) and protected mice from death or emaciation (≥20%) when challenged with widespread influenza virus strains (Figure 2B). Hemagglutinin inhibitory (HAI) titers are reported as log10 for serum samples collected on test days 0, 14, 28, 42, 56, 92, and 107. Bars represent geometric mean and geometric standard deviation. Daily body weight was measured after intranasal challenge with 4LD50 of A / Belgium / 2009(H1N1)(Belgium09) (day 93). Body weight is presented as a percentage of weight loss from the day of challenge. Mice that lost more than 20% of their starting body weight, and all mice euthanized 14 days after infection (day 107). rHA: Recombinant hemagglutinin. AF03: Oil-in-water emulsion adjuvant. Diluent = PBS. LLOQ = Limit of quantification. 1 / 40 = 1 / 40 minimum target, which is the HAI antibody titer associated with a 50% reduction in the risk of influenza infection or disease in healthy adults (Coudeville et al., BMC Med Res Methodol. (2010) 10:18). Dashed line in Figure 2B = 20% body weight loss cropped with respect to body weight on the day of the challenge. [Figure 2B]Figures 2A and 2B are a pair of graphs showing that lipid A and lipid B LNP preparations containing hemagglutinin (HA)-encoding mRNA of strain A / California / 7 / 2009(H1N1)(CA09) induced robust functional antibodies (Figure 2A) and protected mice from death or emaciation (≥20%) when challenged with widespread influenza virus strains (Figure 2B). Hemagglutinin inhibitory (HAI) titers are reported as log10 for serum samples collected on test days 0, 14, 28, 42, 56, 92, and 107. Bars represent geometric mean and geometric standard deviation. Daily body weight was measured after intranasal challenge with 4LD50 of A / Belgium / 2009(H1N1)(Belgium09) (day 93). Body weight is presented as a percentage of weight loss from the day of challenge. Mice that lost more than 20% of their starting body weight, and all mice euthanized 14 days after infection (day 107). rHA: Recombinant hemagglutinin. AF03: Oil-in-water emulsion adjuvant. Diluent = PBS. LLOQ = Limit of quantification. 1 / 40 = 1 / 40 minimum target, which is the HAI antibody titer associated with a 50% reduction in the risk of influenza infection or disease in healthy adults (Coudeville et al., BMC Med Res Methodol. (2010) 10:18). Dashed line in Figure 2B = 20% body weight loss cropped with respect to body weight on the day of the challenge. [Figure 3A]Figures 3A and 3B are a pair of graphs showing that A / Michigan / 45 / 2015 (Mich15) neuraminidase (NA) mRNA formulated with lipid A LNP induced robust functional antibodies (Figure 3A) and protected mice from weight loss and death when challenged with widespread strains of influenza virus (Figure 3B). Neuraminidase inhibitory (NAI) titers are reported as log10 for serum samples collected on study days 14, 28, 42, 56, 88, and 114. Daily body weight was measured after intranasal challenge with 4LD50 Belgium 09 (day 89 for the single-dose group or day 117 for the double-dose group). Body weight is presented as a percentage of weight loss from the challenge day. Euthanasia occurred in mice that experienced a weight loss exceeding 20% ​​of their starting weight, and in all mice 14 days after infection (day 103 in the single-dose group or day 131 in the double-dose group). Bars represent the mean and standard deviation. Upper dashed line in Figure 3A = upper limit of quantification. Lower dashed line in Figure 3A = lower limit of quantification. Dashed line in Figure 3B = 20% weight loss clipped with respect to weight on the challenge day. Administered mRNA: 0.4 or 0.016 μg of mRNA encoding Mich15 NA. Control: 0.6 μg of mRNA or dilution (PBS) encoding hEPO. [Figure 3B]Figures 3A and 3B are a pair of graphs showing that A / Michigan / 45 / 2015 (Mich15) neuraminidase (NA) mRNA formulated with lipid A LNP induced robust functional antibodies (Figure 3A) and protected mice from weight loss and death when challenged with widespread strains of influenza virus (Figure 3B). Neuraminidase inhibitory (NAI) titers are reported as log10 for serum samples collected on study days 14, 28, 42, 56, 88, and 114. Daily body weight was measured after intranasal challenge with 4LD50 Belgium 09 (day 89 for the single-dose group or day 117 for the double-dose group). Body weight is presented as a percentage of weight loss from the challenge day. Euthanasia occurred in mice that experienced a weight loss exceeding 20% ​​of their starting weight, and in all mice 14 days after infection (day 103 in the single-dose group or day 131 in the double-dose group). Bars represent the mean and standard deviation. Upper dashed line in Figure 3A = upper limit of quantification. Lower dashed line in Figure 3A = lower limit of quantification. Dashed line in Figure 3B = 20% weight loss clipped with respect to weight on the challenge day. Administered mRNA: 0.4 or 0.016 μg of mRNA encoding Mich15 NA. Control: 0.6 μg of mRNA or dilution (PBS) encoding hEPO. [Figure 4] Figure 4 is a graph showing that lipid A and lipid B LNP preparations (10 μg) containing CA09 HA mRNA induced robust functional antibodies in cynopaks. HAI titers are reported as log2 for serum samples collected on test days 0, 14, 28, 42, and 56. [Figure 5A-1] Figures 5A-5C show the MRT1400 mRNA encoding influenza A / Singapore / INFIMH160019 / 2016(Sing16;H3N2)HA hemagglutinin. Figure 5A: Alignment of the wild-type (WT) gene and codon-optimized gene (MRT10279) for the HA antigen. Figure 5B: mRNA structure. Figure 5C: mRNA sequence. [Figure 5A-2] Continuation of Figure 5A-1. [Figure 5A-3] Continuation of Figure 5A-2. [Figure 5A-4] Continuation of Figure 5A-3. [Figure 5A-5] Continuation of Figure 5A-4. [Figure 5B] Figures 5A-5C show the MRT1400 mRNA encoding influenza A / Singapore / INFIMH160019 / 2016(Sing16;H3N2)HA hemagglutinin. Figure 5A: Alignment of the wild-type (WT) gene and codon-optimized gene (MRT10279) for the HA antigen. Figure 5B: mRNA structure. Figure 5C: mRNA sequence. [Figure 5C] Figures 5A-5C show the MRT1400 mRNA encoding influenza A / Singapore / INFIMH160019 / 2016(Sing16;H3N2)HA hemagglutinin. Figure 5A: Alignment of the wild-type (WT) gene and codon-optimized gene (MRT10279) for the HA antigen. Figure 5B: mRNA structure. Figure 5C: mRNA sequence. [Figure 6] Figure 6 shows a pair of graphs illustrating that lipid A and lipid B LNP preparations containing MRT1400 or NA mRNA induced robust functional antibodies in mice. The first injection was administered on test day 0, and the second injection on test day 28. Left panel: HAI titers are reported as log10 for serum samples collected on test days 14, 28, 42, and 56. Right panel: NAI titers are reported as log10 for serum samples collected on test days 14, 28, 42, and 56. Bars represent the geometric mean and geometric standard deviation. Dashed line = limit of quantification. [Figure 7A] Figure 7A is a graph showing that lipid A and lipid B LNP preparations with MRT1400 induced robust functional antibodies in NHP. HAI titers are reported as log2 for serum samples collected on test days 0, 14, 28, 42, and 56. The first injection was administered on test day 0, and the second injection on test day 28. The bars represent the mean and standard deviation. The upper dashed line = 1 / 40 minimum target. The lower dashed line = lower limit of detection. [Figure 7B]Figures 7B and 7C are a pair of figures showing that a lipid A LNP preparation (MRT5400) containing MRT1400 mRNA induced functional antibodies (Figure 7B) and robust ELISA titers (Figure 7C) in cynophobic macaques at four dose levels of mRNA: 15, 45, 135, and 250 μg. HAI and ELISA titers are reported as log2 for serum samples collected on test days 0, 14, 28, 42, and 56. The first injection was given on test day 0, and the second injection was given on test day 28. Bars represent the mean and standard deviation. Dashed line = 1 / 40 minimum target. [Figure 7C] Figures 7B and 7C are a pair of figures showing that a lipid A LNP preparation (MRT5400) containing MRT1400 mRNA induced functional antibodies (Figure 7B) and robust ELISA titers (Figure 7C) in cynophobic macaques at four dose levels of mRNA: 15, 45, 135, and 250 μg. HAI and ELISA titers are reported as log2 for serum samples collected on test days 0, 14, 28, 42, and 56. The first injection was given on test day 0, and the second injection was given on test day 28. Bars represent the mean and standard deviation. Dashed line = 1 / 40 minimum target. [Figure 8A] Figures 8A and 8B are panels of graphs showing the T cell cytokine response in cynocephalic macaques after a second vaccination with the lipid A LNP preparation MRT5400 at three dose levels (250 μg, 135 μg, and 45 μg mRNA). IFN-γ and IL-13 induced by restimulation with recombinant HA (rHA) protein (left panel) or pooled peptide (right panel) were evaluated in peripheral blood mononuclear cells (PMBCs) at day 42 by the ELISPOT assay. The incidence of PBMC-secreted IFN-γ (Figure 8A) or IL-13 (Figure 8B) was calculated as spotting cells (SFCs) per million PBMCs. Each symbol represents an individual sample, and the bar represents the standard deviation. [Figure 8B]Figures 8A and 8B are panels of graphs showing the T cell cytokine response in cynocephalic macaques after a second vaccination with the lipid A LNP preparation MRT5400 at three dose levels (250 μg, 135 μg, and 45 μg mRNA). IFN-γ and IL-13 induced by restimulation with recombinant HA (rHA) protein (left panel) or pooled peptide (right panel) were evaluated in peripheral blood mononuclear cells (PMBCs) at day 42 by the ELISPOT assay. The incidence of PBMC-secreted IFN-γ (Figure 8A) or IL-13 (Figure 8B) was calculated as spotting cells (SFCs) per million PBMCs. Each symbol represents an individual sample, and the bar represents the standard deviation. [Figure 9A] Figure 9A is a pair of graphs showing that lipid A LNP preparations containing modified and unmodified CA09 HA mRNA were comparable to those indicated by HAI titers in vaccinated mice. HAI titers are reported as log2 for serum samples collected on test days 14, 28, 42, and 56. The first injection was given on test day 0, and the second injection on test day 28. The bars represent the mean and standard deviation. The upper dashed line = 1 / 40 minimum target. The lower dashed line = lower limit of quantification. [Figure 9B] Figure 9B is a pair of graphs showing that lipid A LNP preparations containing modified and unmodified CA09 HA mRNA were comparable to those indicated by ELISA titers in mice. Total IgG ELISA titers are reported as log10 for serum samples collected on test days 14, 28, 42, and 56. The first injection was administered on test day 0, and the second injection on test day 28. Dashed line = limit of quantification. [Figure 10]Figures 10A and 10B are a pair of graphs showing that divalent lipid A LNP preparations containing CA09 HA mRNA and Sing16 HA mRNA induced robust functional antibodies in Balb / c mice when evaluated by HAI titer (CA09 (Figure 10A) and Sing16 (Figure 10B)) at a dose of 0.4 μg of total mRNA. 0.4 μg of mRNA was administered as a co-encapsulated mRNA-LNP preparation, or each HA mRNA was administered separately, with 0.2 μg in each lower limb. Each HA mRNA was also co-encapsulated in the preparation with non-coding mRNA to control that the total mRNA was packed into the LNP. Dilution groups received mRNA-LNP dilution buffer. HAI titers are reported for serum samples collected on test days 2 (baseline), 14, 28, and 42. Figure 10B shows only test days 2 (baseline from pooled serum) and 42. The first injection was administered on test day 0, and the second injection was administered on test day 28. The bars represent the geometric mean and geometric standard deviation. The dashed line = lower limit of quantification. [Figure 11]Figure 11 shows the functional validation of mRNA-LNP preparations. Panel (a) is a graph showing the expression of firefly (FF) luciferase in BALB / c mice: single doses of luciferase FF mRNA-LNP (5, 1, 0.1, 0.05 μg) were injected into mice (n=4) via the IM pathway. Luciferin (3 mg) was injected during whole-body imaging using IVIS Spectrum, Perkin Elmer, to record bioluminescence intensity. Whole-body mean luminescence images were taken at 6, 24, 48, and 72 hours after injection. Recorded luminescence for Luc mRNA-LNP doses of 1, 0.5, 0.1, and 0.05 μg is shown in the graph. Panel (b) shows whole-body images showing the total luminescence flux at 6–72 hours. The total luminescence flux in the group of mice (n=4) receiving a 0.1 μg dose of FF-LNP is shown. Panel (c) shows hEPO expression in BALB / c mice. A single dose of hEPO mRNA-LNP (0.1 μg) was injected via the IM route into BALB / c mice. hEPO expression was quantified in serum using ELISA at 6 and 24 hours post-administration. Bars represent the mean and standard deviation. Panel (d) shows hEPO expression in NHP. A single dose of hEPO mRNA-LNP (10 μg) was injected via the IM route into cynophore macaques. hEPO expression was quantified in serum using ELISA at 6, 24, 48, 72, and 96 hours post-administration. Bars represent the mean and standard deviation. [Figure 12] Figure 12 shows the serological evaluation of HA mRNA-LNP vaccines in mice. BALB / c mice (n=8 per group) were twice immunized with IM using Cal09 HA mRNA-LNP or Sing16 HA mRNA-LNP at 2, 0.4, 0.08, and 0.016 μg, with a 4-week interval between doses. ELISA titers recorded for serum collected on days 14, 28, 42, and 56 against CA09 (Cal09) H1N1 influenza virus recombinant HA (left panel) and Sing16 H3N2 influenza virus recombinant HA (right panel) are shown. [Figure 13]Figure 13 shows the serological evaluation of HA mRNA-LNP vaccines in mice. BALB / c mice (n=8 per group) were twice immunized with IM using CA09 HA mRNA-LNP or Sing16 HA mRNA-LNP at 2, 0.4, 0.08, and 0.016 μg, with a 4-week interval between doses. Log10HAI titers recorded against CA09 H1N1 influenza virus (left panel) and Sing16 H3N2 influenza virus (right panel) are shown. [Figure 14] Figure 14 shows the serological evaluation of NA mRNA-LNP vaccines in mice. BALB / c mice (n=8 per group) were twice immunized with IM with Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP at 2, 0.4, 0.08, and 0.016 μg, with a 4-week interval between doses. Total IgG titers recorded for serum collected on days 0, 14, 28, 42, and 56 against Mich15 N1 influenza virus recombinant NA (left panel) and Sing16 N2 influenza virus recombinant NA (right panel) are shown. [Figure 15] Figure 15 shows the serological evaluation of NA mRNA-LNP vaccines in mice. BALB / c mice (n=8 per group) were twice immunized with IM using Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP at 2, 0.4, 0.08, and 0.016 μg, with a 4-week interval between doses. Log10NAI(ELLA) titers recorded for serum against Mich2015(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus (left panel) and Sing16(N2):A / Mallard / Sweden / 2002(H6) chimeric virus (right panel) are shown. [Figure 16]Figures 16A and 16B show the protective efficiency of the CA09 HA mRNA-LNP vaccine in mice after lethal A / Belgium / 2009 H1N1 virus challenge. Mice (n=8) received two doses of CA09 HA mRNA-LNP (0.4 μg each) on day 0 and day 28. Control animals received two doses of diluted IM on day 0 and day 28. Figure 16A shows HAI titers reported as Log10 for serum samples collected on test days 0, 14, 28, 42, 56, 92, and 107. Figure 16B shows daily body weight after intranasal challenge on day 93 with 4LD50 A / Belgium / 2009 H1N1 strain. Body weight is presented as a percentage of weight loss from the day of challenge. Individual lines represent each animal. [Figure 17A] Figures 17A-B show the protective efficiency of single-dose unmodified Mich15 NA mRNA-LNP in mice after lethal A / Belgium / 2009 H1N1 virus challenge. Mice (n=16) were injected with 0.4 μg or 0.016 μg of Mich15 NA mRNA-LNP via the IM route. Half of the mice received only one injection (1 dose) on test day 0, and the other half (2 doses) received two injections on test day 0 and test day 28. Control animals received two IM doses (0.6 μg) of hEPO mRNA-LNP on day 0 and day 28. Figure 17A shows that NAI titers are reported as Log10 for serum samples collected on test days 0, 14, 28, 42, 56, 88, and 114. Figure 17B shows the daily weight changes after an intranasal challenge using 4LD50 Belgium 09 H1N1 in the single-dose group at day 89 and in the double-dose group at day 117. Weight is presented as a percentage of weight loss from the day of the challenge. Each line represents a different animal. [Figure 17B]Figures 17A-B show the protective efficiency of single-dose unmodified Mich15 NA mRNA-LNP in mice after lethal A / Belgium / 2009 H1N1 virus challenge. Mice (n=16) were injected with 0.4 μg or 0.016 μg of Mich15 NA mRNA-LNP via the IM route. Half of the mice received only one injection (1 dose) on test day 0, and the other half (2 doses) received two injections on test day 0 and test day 28. Control animals received two IM doses (0.6 μg) of hEPO mRNA-LNP on day 0 and day 28. Figure 17A shows that NAI titers are reported as Log10 for serum samples collected on test days 0, 14, 28, 42, 56, 88, and 114. Figure 17B shows the daily weight changes after an intranasal challenge using 4LD50 Belgium 09 H1N1 in the single-dose group at day 89 and in the double-dose group at day 117. Weight is presented as a percentage of weight loss from the day of the challenge. Each line represents a different animal. [Figure 18] Figure 18 shows the serological evaluation of the HA Sing16 HA mRNA-LNP vaccine in NHP. Crab-eating macaques (n=6 per group) were injected twice via the IM route with 15, 45, or 135 μg of Sing16 HA mRNA-LNP, 4 weeks apart. Serum samples were collected on days 6, 14, 28, 42, and 56. Log10 IgG titers against recombinant HA protein of the Sing16 virus are shown. [Figure 19] Figures 19A and 19B show the serological evaluation of the HA Sing16 HA mRNA-LNP vaccine in NHP. Cyanobacteria macaques (n=6 per group) were injected twice via the IM route with 15, 45, or 135 μg of Sing16 HA mRNA-LNP, 4 weeks apart. Serum samples were collected on days 0, 14, 28, 42, and 56. Log10 HAI titers (Figure 19A) and Log10 microneutralizing (MN) titers (Figure 19B) against the Sing16 virus are shown. [Figure 20]Figures 20A and 20B show the T cell response in NHP vaccinated with the Sing16 HA mRNA-LNP vaccine. Cyanobacteria macaques (n=6 per group) were injected twice via the IM route with 45, 135, or 250 μg of Sing16 HA mRNA-LNP, 4 weeks apart. T cells were assessed by ELISPOT on day 42 in PBMCs stimulated in vitro using a peptide pool representing the entire HA open reading frame. The response of PBMCs secreting IFN-γ (Figure 20A) or IL-13 (Figure 20B) is shown, calculated as spot-forming cells (SFCs) per million PBMCs. Each symbol represents an individual sample, and the bars represent the geometric mean for the group. [Figure 21] Figure 21 shows the secretion of Sing16 H3-specific IgG by memory B cells at day 180 in NHP vaccinated with Sing16 HA mRNA-LNP vaccine. Cyanobacteria macaques (n=6 per group) were injected twice via the IM route with 15 or 45 μg of Sing16 HA mRNA-LNP, 4 weeks apart. Sing16 / H3-specific and total IgG+ antibody-secreting cells (ASCs) were measured using the Human IgG Monochromatic Memory B Cell ELISPOT Kit (CAT# NC1911372, CTL). Differentiation of MBCs into ASCs was performed in PBMCs collected at day 180 using the stimulation cocktail provided by the kit. The number of IgG+ cells and Sing16 / H3-specific ASCs were calculated per million PBMCs for each animal, and the frequency of antigen-specific ASC occurrence is shown. [Figure 22-1]Figure 22 shows the delivery of bivalent combination influenza vaccines in mice. BALB / c mice (n=8 per group) were immunized twice with IM at 4-week intervals, either with a total of 0.4 μg of the bivalent combination co-encapsulated mRNA transcript (1:1 wt / wt, half a dose per leg) or with 0.2 μg of each monovalent vaccine, separately formulated and immunized in different legs. CA09 HA mRNA-LNP and Sing16 HA mRNA-LNP, which constitute the H1H3 combo; Sing16 HA mRNA-LNP and Sing16 NA mRNA-LNP, which constitute the H3N2 combo; and Mich15 NA mRNA-LNP and Perth09 NA mRNA-LNP, which constitute the N1N2 combo, were tested against the corresponding viruses in serum collected on days 0, 14, 28, and 42. Panel (a) shows the HAI titers recorded against CA09 H1N1 influenza virus and Sing2016 H3N2. Panel (b) shows the HAI and NAI titers recorded for Sing2016 H3N2 and A / Mallard / Sweden / 2002(H6) chimeric influenza viruses and H6N2 A / Perth / 09 virus F1919D(N2), respectively. Panel (c) shows the NAI titers recorded for Mich15(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus and H6N2 A / Perth / 09 virus F1919D(N2), respectively. [Figure 22-2] Continuation of Figure 22-1. [Figure 23]Figure 23 shows the delivery of quadrivalent combinations of influenza vaccine in NHP. Crab-eating macaques (n=6 per group) were immunized twice with 10 μg of the total quadrivalent combination (1:1:1:1 wt / wt) of co-encapsulated mRNA transcripts with an interval of 4 weeks between doses. H2H3N1N2 combo consisting of CA09 HA mRNA, Sing16 HA mRNA, Mich15 NA mRNA, and Perth09 NA mRNA; H1H3 combo consisting of CA09 HA mRNA, Sing16 HA mRNA, and 2× non-coding mRNA (ncmRNA); H3N2 combo of Sing16 HA mRNA, Perth09 NA mRNA, and 2× non-coding mRNA; N1N2 combo of Mich15 NA mRNA, Perth09 NA mRNA-LNP, and 2× non-coding mRNA; H1 combo consisting of CA09 HA mRNA and 3× non-coding mRNA. H3 consists of Sing16 HA mRNA and 3× non-coding mRNA. N1 consists of Mich15 NA mRNA and 3× non-coding mRNA. N2 consists of Perth09 NA mRNA and 3× non-coding mRNA. Inhibitory titers were tested against the corresponding viruses in serum collected on days 0, 14, 28, and 42. Panel (a) shows the HAI titers recorded against CA09 H1N1 influenza virus and Sing16 H3N2. Panel (b) shows the NAI titers recorded against Mich15(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus and H6N2 Perth / 09 virus F1919D(N2) virus. [Figure 24] Figure 24 shows a graph illustrating the expression of human erythropoietin (hEPO) mRNA in mice treated with various LNP formulations. The LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. The bars represent the mean and standard deviation. The LNP compositions contain cationic lipids, DMG-PEG2000, cholesterol, and DOPE in a molar ratio of 40:1.5:28.5:30, in that order. [Figure 25]Figure 25 shows a graph illustrating the expression of hEPO mRNA in non-human primates (NHPs) treated with various LNP formulations. The LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. The bars represent the mean and standard deviation. The LNP compositions contain cationic lipids, DMG-PEG2000, cholesterol, and DOPE in a molar ratio of 40:1.5:28.5:30, in that order. [Figure 26] Figure 26 shows graphs of HAI titers at 28 and 42 days post-injection for various LNP formulations of HA mRNA. The LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. The bars represent the mean and standard deviation. The LNP compositions contain cationic lipids, DMG-PEG2000, cholesterol, and DOPE in a molar ratio of 40:1.5:28.5:30, in that order. [Figure 27] Figure 27 shows graphs of CalO9 H1 HAI titers at 28 and 42 days post-injection for various LNP formulations of HA mRNA. The LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. The bars represent the mean and standard deviation. The LNP compositions contain cationic lipids, DMG-PEG2000, cholesterol, and DOPE in a molar ratio of 40:1.5:28.5:30, in that order. [Figure 28] Figure 28 shows graphs of Sing16 H3 HAI titers at 28 and 42 days post-injection for various LNP formulations of HA mRNA. The LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. The bars represent the mean and standard deviation. The LNP compositions contain cationic lipids, DMG-PEG2000, cholesterol, and DOPE in a molar ratio of 40:1.5:28.5:30, in that order. [Figure 29]Figure 29 shows the RSV F protein antibody titer in NHP immunized with FD3 F protein expression mRNA. mRNA was delivered via lipid nanoparticles (LNPs) containing one of several cationic lipids. Antibody titers were measured for each antigen composition at days 0, 21, and 35. [Figure 30] Figure 30 shows the RSV neutralizing titer in NHP immunized with FD3 F protein-expressing mRNA. mRNA was delivered via lipid nanoparticles (LNPs) containing one of several cationic lipids. Antibody titers were measured for each antigen composition at days 0, 21, and 35. [Figure 31] Figure 31 shows the HAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to mice against four different influenza strains. [Figure 32] Figure 32 shows the HINT values ​​for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. [Figure 33] Figure 33 shows the NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to mice against four different influenza strains. [Figure 34] Figure 34 shows the NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. Samples were obtained 20 days (D20) after the second dose of the vaccine. [Figure 35] Figure 35 shows the NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. Samples were obtained 42 days (D42) after the second dose of the vaccine. [Figure 36] Figure 36 shows the microneutralization titers for Sing16HA-coding mRNA in lipid A LNP preparations administered to NHP at doses of 15 μg and 45 μg. Samples were obtained on day 6 (D6) and day 42 (D42) after the second dose of the vaccine. [Figure 37]Figure 37 shows the microneutralization titers for Sing16HA-coding mRNA in lipid B LNP preparations administered to NHP at doses of 15 μg and 45 μg. Samples were obtained on day 6 (D6) and day 42 (D42) after the second dose of the vaccine. [Modes for carrying out the invention]

[0034] This disclosure provides novel lipid nanoparticle (LNP) formulations and methods for producing vaccines for delivering mRNA vaccines in vivo. LNPs are made from a mixture of four lipids: cationic lipids, polyethylene glycol (PEG) conjugated lipids, cholesterol-based lipids, and helper lipids. LNPs encapsulate mRNA molecules. The encapsulated mRNA molecules may contain naturally occurring ribonucleotides, chemically modified nucleotides, or combinations thereof, each or collectively encoding one or more proteins.

[0035] The inventors discovered this formulation by screening a combinatorial library of lipid components. This LNP encapsulates the mRNA payload, protecting it from degradation and promoting the intracellular uptake of the encapsulated mRNA. Compared to industrial formulations described herein, the LNP described herein enhances transduction efficiency and promotes mRNA extrusion into endosomes, as demonstrated by enhanced expression in vivo and in vitro, resulting in improved efficacy. For example, the LNPs disclosed herein have superior stability and / or efficacy profiles compared to known LNPs, such as heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoic acid (aka DLin-MC3-DMA or MC3; Semple et al., Nat Biotechnol. (2010) 28: pp. 172-176) or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanoic acid (aka L319; Maier et al., Mol Ther. (2013) 21(8): pp. 1570-158). As further described below, the formulations encapsulating the hEPO-encoding mRNA, when delivered in vivo, result in high levels of erythropoietin circulating in the blood at 6 and 24 hours, up to a 12-fold increase compared to the industrial standard, MC3 LNP formulation. Similarly, high efficacy was observed with other mRNAs, such as the mRNA encoding the influenza antigen, in both mouse and non-human primate models.

[0036] The mRNA vaccines formulated herein can induce a balanced immune response encompassing both cellular and humoral immunity. The advantage of these LNP formulations is their non-sequence-specific nature; therefore, they can deliver mRNA encoding a variety of antigens, enabling rapid deployment in epidemic or pandemic situations. Furthermore, these LNP-formulated mRNA vaccines are highly immunogenic, thus offering significant dose-saving potential.

[0037] I. Composition of these lipid nanoparticles This LNP includes four categories of lipids: (i) ionizable lipids, (ii) pegylated lipids, (iii) cholesterol-based lipids, and (iv) helper lipids.

[0038] A. Ionizable lipids Ionizable lipids promote mRNA encapsulation, and cationic lipids may also be used. Cationic lipids provide a positively charged environment at low pH, promoting the efficient encapsulation of negatively charged mRNA drug substances.

[0039] In some embodiments, the cationic lipid is OF-02.

[0040] [ka] OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains a degradable ester bond for binding to a diketopiperazine core and a biunsaturated tail, while OF-02 contains a non-degradable 1,2-amino alcohol bond for binding to the same diketopiperazine core and biunsaturated tail (Fenton et al., Adv Mater. (2016) 28:2939; U.S. Patent No. 10,201,618). (No.). The exemplary LNP formulations described herein, lipid A contains OF-2.

[0041] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11): pp. 3955-3960; U.S. Patent No. 9,512,073).

[0042] [ka] The exemplary LNP formulations described herein, lipid B, contain cKK-E10.

[0043] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1(2-(4-(2-((3-(bis((Z)-2-hydroxyoctadeca-9-en-1-yl)amino)propyl)disulfaneyl)ethyl)piperazine-1-yl)ethyl4-(bis(2-hydroxydecyl)amino)butanoic acid, which is a HEPES-based disulfide cationic lipid having a piperazine core and having formula (III).

[0044] [ka] In the exemplary LNP formulations described herein, lipid C contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C has the same composition as lipid A or lipid B, except for the difference in cationic lipids.

[0045] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10(2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl) Disulfaneyl)ethyl)piperazine-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoic acid is a HEPES-based disulfide cationic lipid having a piperazine core and having formula (IV).

[0046] [ka] In the exemplary LNP formulations described herein, lipid D contains GL-HEPES-E3-E12-DS-4-E10. Lipid D has the same composition as lipid A or lipid B, except for the difference in cationic lipids.

[0047] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14(2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazine-1-yl)ethyl4-(bis(2-hydroxydodecyl)amino)butanoic acid, which is a HEPES-based disulfide cationic lipid having a piperazine core and having formula (V).

[0048] [ka] In the exemplary LNP formulations described herein, lipid E ​​contains GL-HEPES-E3-E12-DS-3-E14. Lipid E ​​has the same composition as lipid A or lipid B, except for the difference in cationic lipids.

[0049] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1(III), GL-HEPES-E3-E12-DS-4-E10(IV), and GL-HEPES-E3-E12-DS-3-E14(V) can be synthesized according to the general procedure shown in Figure 1.

[0050] Figure 1: General synthesis diagrams for lipids of formulas (III), (IV), and (V) [ka]

[0051] Other cationic lipids that can be used include those described in Dong, see above, and U.S. Patent No. 10,201,618.

[0052] B. Pegylated lipids Pegylated lipid components control the particle size and stability of nanoparticles. Adding such components can provide a means to prevent complex aggregation, increase circulating lifespan, and enhance delivery of lipid-nucleic acid drug compositions to target tissues (Klibanov et al., FEBS Letters (1990) 268(1): pp. 235-237). These components can be selected for rapid replacement from the drug composition in vivo (see, e.g., U.S. Patent No. 5,885,613).

[0053] The intended PEGylated lipid is a derivatized ceramide (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)](C8 PEG ceramide) )) C6~C 20 (For example, C8, C 10 , C 12 , C 14 , C 16 , or C 18 The PEGylated lipid includes, but is not limited to, polyethylene glycol (PEG) with a maximum length of 5 kDa covalently bonded to a lipid having an alkyl chain (or more) of the )(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).

[0054] In particularly exemplary embodiments, PEG has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, PEG is PEG2000 (or PEG-2K). In specific embodiments, the PEGylated lipids herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000.

[0055] C. Cholesterol lipids The cholesterol component stabilizes 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), 1,4-bis(3-N-aleylaminopropyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol ester ("ICE"; WO2011 / 068810), β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0056] D. Helper lipids Helper lipids enhance the structural stability of LNPs and facilitate LNP endosomal escape. Helper lipids improve the uptake and release of mRNA drug payloads. In some embodiments, the helper lipids are zwitterionic lipids, which have membrane fusion properties to enhance drug payload uptake and release. 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), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-distearoylphosphatidylethanolamine (DSPE); and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0057] Other exemplary helper lipids include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 1 These are 6-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a combination thereof.

[0058] In certain embodiments, the helper lipid is DOPE. In further 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.

[0059] E. Molar ratio of lipid components The inventors have discovered that specific molar ratios of the above components are important for the effectiveness of LNPs in delivering mRNA. 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 the LNP compared to total lipids (i.e., A) is 35-45% (e.g., 38-42%, such as 40%). In some embodiments, the molar ratio of pegylated lipid components compared to total lipids (i.e., B) is 0.25-2.75% (e.g., 1-2%, such as 1.5%). In some embodiments, the molar ratio of cholesterol lipids compared to total lipids (i.e., C) is 20-35% (e.g., 27-30%, such as 28.5%). In some embodiments, the molar ratio of helper lipids to total lipids (i.e., D) is 25-35% (e.g., 28-32%, such as 30%). In some embodiments, the (pegylated lipid + cholesterol) component has the same molar amount as the helper lipids. In some embodiments, the LNP contains a cationic lipid to helper lipid molar ratio greater than 1.

[0060] In certain embodiments, LNP contains cationic lipids, pegylated lipids, cholesterol-based lipids, and helper lipids in a molar ratio of 40:1.5:28.5:30. In further specific embodiments, LNP contains (i) 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 in a ratio of 40:1.5:28.5:30.

[0061] To calculate the actual amount of each lipid to be included in the LNP formulation, the molar amount of the cationic lipid is first determined based on the desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the selected molar ratio. Then, these molar amounts are converted to weight using the molecular weight of each lipid.

[0062] Active components of F.LNP The active ingredient of this LNP vaccine composition is mRNA encoding the target antigen. The antigen may be a polypeptide derived from a virus, such as influenza virus, coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, or MERS-related virus), Ebola virus, dengue virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), polynuclear respiratory virus (RSV), rhinovirus, cytomegalovirus (CMV), Zika virus, human papillomavirus (HPV), human metapneumovirus (hMPV), human parainfluenza virus type 3 (PIV3), Epstein-Barr virus (EBV), chikungunya virus, or polynuclear respiratory virus (RSV).

[0063] The antigens include bacteria such as Staphylococcus aureus, Moraxella (e.g., Moraxella catarrhalis; which causes otitis, respiratory infections, and / or sinusitis), Chlamydia trachomatis (which causes chlamydia), Borrelia (e.g., Borrelia burgdorferi; which causes Lyme disease), Bacillus anthracis (which causes anthrax), Salmonella typhi (which causes typhoid fever), Mycobacterium tuberculosis (which causes tuberculosis), and Propionibacterium acnes. It may be derived from acne (causing lacerations) or from non-typeable Haemophilus influenzae.

[0064] If desired, LNPs or LNP formulations may be polyvalent. In some embodiments, LNPs may harbor mRNAs encoding more than one antigen, such as two, three, four, five, six, seven, eight, nine, ten, or more antigens from the same or different pathogens. For example, an LNP may harbor multiple mRNA molecules, each encoding a different antigen; or it may harbor polycistronic mRNA capable of translating to more than one antigen (e.g., each antigen-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide, such as a 2A peptide). LNPs harboring different mRNA molecules typically contain (encapsulate) multiple copies of each mRNA molecule. For example, an LNP harboring or encapsulating two different mRNA molecules typically contains multiple copies of each of the two different mRNA molecules.

[0065] In some embodiments, a single LNP formulation may contain multiple types (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of LNPs, each possessing a different mRNA.

[0066] An example of a polyvalent LNP vaccine is an LNP vaccine containing mRNA encoding two or more antigens from the above-mentioned pathogen, such as an LNP vaccine containing mRNA encoding a polypeptide derived from the influenza virus. In some embodiments, the polyvalent LNP vaccine contains mRNA molecules encoding polypeptides derived from two or more (e.g., three, four, five, six, seven, eight, nine, or ten) influenza virus proteins 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 polyvalent LNP vaccine contains two or more (e.g., three, four, five, six, seven, eight, or more) mRNA molecules encoding antigenic polypeptides derived from HA protein, NA protein, and both HA and NA proteins. In some embodiments, the mRNA molecules encoding the antigenic polypeptide are derived from different influenza strains.

[0067] In certain embodiments, the composition may contain one or more mRNA molecules encoding antigens of influenza A, B, and C viruses. In one embodiment, the composition may contain one or more mRNA molecules encoding HA and / or NA antigens of influenza A and influenza B viruses. In one embodiment, 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 one embodiment, the NA antigen of influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. In one embodiment, the HA and NA antigens of influenza B virus are derived from the influenza B / Yamagata lineage. In one embodiment, the HA and NA antigens of influenza B virus are derived from the influenza B / Victoria lineage. In some embodiments, one or more HA and NA antigens are derived from influenza virus strains recommended by the World Health Organization (WHO) in its annual recommendations for influenza vaccine formulations.

[0068] In a particular embodiment, at least one of one or more influenza virus proteins comprises an influenza virus HA protein and / or influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and 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.

[0069] In certain embodiments, the composition comprises (i) one or more HA antigens, (ii) one or more NA antigens, or (iii) two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding one or more combinations of HA antigens and NA antigens.

[0070] In one embodiment, the composition comprises two, three, four, five, six, seven, eight, nine, or more mRNA molecules, selected from the H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H10N7 subtypes and / or B / Yamagata and B / Victoria lineages, encoding i) one or more HA antigens, (ii) one or more NA antigens, or (iii) one or more combinations of HA antigens and NA antigens.

[0071] In one embodiment, the composition comprises one mRNA molecule encoding an H3 HA antigen, one mRNA molecule encoding an H1 HA antigen, one mRNA molecule encoding an HA antigen from the influenza B / Yamagata lineage, and one mRNA molecule encoding an HA antigen from influenza B / Victoria.

[0072] In one embodiment, the composition comprises one mRNA molecule encoding the H3 HA antigen, one mRNA molecule encoding the N2 NA antigen, one mRNA molecule encoding the H1 HA antigen, one mRNA molecule encoding the N1 NA antigen, one mRNA molecule encoding the HA antigen from the influenza B / Yamagata lineage, one mRNA molecule encoding the NA antigen from the influenza B / Yamagata lineage, one mRNA molecule encoding the HA antigen from the influenza B / Victoria lineage, and one mRNA molecule encoding the NA antigen from the influenza B / Victoria lineage.

[0073] In embodiments, the composition further comprises one or more mRNA molecules encoding a machine learning influenza virus HA having a molecular sequence identified or designed from a machine learning model, wherein one or more machine learning influenza virus HAs are H1 You may choose from HA, H3 HA, HA derived from the B / Victoria lineage, HA derived from the B / Yamagata lineage, or a combination thereof.

[0074] When selecting one or more machine learning influenza virus HAs, Machine learning algorithms may be used. For example, either of the machine learning algorithms and methods disclosed in PCT application International Publication 2021 / 080990, titled "Systems and Methods for Designing Vaccines," and PCT application International Publication 2021 / 080999, titled "Systems and Methods for Predicting Biological Responses," both of which are incorporated herein by reference in their entirety.

[0075] The mRNA molecule may be unmodified (i.e., containing only native ribonucleotides A, U, C, and / or G linked by phosphate diester bonds) or chemically modified (e.g., including nucleotide analogues such as pseudouridine (e.g., N-1-methylpsoiduridine), 2'-fluororibonucleotide, and 2'-methoxyribonucleotide, as well as / or phosphorothioate bonds). The mRNA molecule may also contain a 5' cap and a polyA tail.

[0076] RSV F protein Respiratory multinuclear virus (RSV) is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae. RSV can cause respiratory tract infections. RSV is an enveloped virus and has glycoproteins (G proteins), small hydrophobic proteins (SH proteins), and fusion proteins (F proteins) on its surface.

[0077] The RSV F protein is responsible for the fusion of the viral membrane and the host cell membrane and has at least three conformations (pre-fusion, intermediate, and post-fusion conformations). In the pre-fusion conformation (Pre-F), the F protein exists as a trimer with its major antigen site Φ exposed. Site Φ serves as the primary target of neutralizing antibodies produced by RSV infection targets (see Coultas et al., Thorax. 74: pp. 986-993, 2019; McLellan et al., Science. 340 (6136): pp. 1113-117, 2013). After binding to its target on the host cell surface, Pre-F undergoes a conformational change during which site Φ is no longer exposed. Pre-F transitions to a transient intermediate conformation, allowing the F protein to be inserted into the host cell membrane, and the viral membrane and host cell membrane to fuse. The final conformational shift results in a more stable, elongated shape of the protein (post-fusion, Post-F). Sites II and IV of the F protein are specific to Post-F, while site I is present in both Pre-F and Post-F three-dimensional structures (McLellan et al., J. Virol. 85(15):7788-7796, 2011).

[0078] As used herein, the term “F protein” or “RSV F protein” refers to the RSV protein responsible for driving the fusion of the viral envelope with the host cell membrane during viral entry.

[0079] As used herein, the terms “RSV F polypeptide” or “F polypeptide” mean a polypeptide that contains at least one epitope of the F protein.

[0080] As used herein, the term “post-fusion” with respect to RSV F refers to the stable three-dimensional structure of RSV F that results from the fusion of the virus and the cell membrane.

[0081] As used herein, the term “pre-fusion” with respect to RSV F refers to the three-dimensional structure of RSV F prior to the virus-cell interaction.

[0082] mRNA molecules encoding antigenic RSV F polypeptide are provided herein.

[0083] In some embodiments, the mRNA molecule is the respiratory multinuclear virus (RSV) F protein. It contains an open reading frame (ORF) that codes for an antigen.

[0084] In some embodiments, the RSV F protein antigen includes a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 16.

[0085] In some embodiments, the RSV F protein antigen contains an amino acid sequence that is at least 98% identical to SEQ ID NO: 16, or consists of the amino acid sequence of SEQ ID NO: 16.

[0086] In some embodiments, the mRNA includes a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence shown in SEQ ID NO: 17.

[0087] In some embodiments, the mRNA includes a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence shown in SEQ ID NO: 21.

[0088] In some embodiments, the RSV F protein antigen is a pre-fusion protein.

[0089] In some embodiments, the ORF is codon-optimized.

[0090] In some embodiments, the mRNA molecule includes at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and at least one polyadenylated (poly(A)) sequence.

[0091] In some embodiments, the mRNA contains at least one chemical modification.

[0092] 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.

[0093] 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.

[0094] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpsoiduridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-psoiduridine, 2-thio-1-methylpsoiduridine, 2-thio-5-aza-uridine, 2-thio-dihydropsoiduridine, 2-thio-dihydrouridine, 2-thiopsoiduridine, 4-methoxy-2-thiopsoiduridine, 4-methoxypsoiduridine, 4-thio-1-methylpsoiduridine, 4-thiopsoiduridine, 5-aza-uridine, dihydropsoiduridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0095] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpsoiduridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, the chemical modification is N1-methylpsoiduridine.

[0096] In some embodiments, mRNA includes the following structural elements: (i) A 5' cap having the following structure; [ka] (ii) The 5' untranslated region (5'UTR) having the nucleic acid sequence of sequence number 19; (iii) Protein coding region having the nucleic acid sequence of Sequence ID No. 17; (iv) The 3' untranslated region (3'UTR) having the nucleic acid sequence of sequence number 20; and (v) Poly(A) tail.

[0097] G. Buffer 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 an LNP pharmaceutical composition, and / or to enhance the in vivo expression of nucleic acids, nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants) and / or other pharmaceutically acceptable excipients. Examples of such excipients include parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, chelating agents (e.g., EDTA) and similar.

[0098] The LNP compositions of this disclosure can be provided in a cryogenic liquid form or a lyophilized form. Various cryoprotective substances may be used, including but not limited to sacrose, trehalose, glucose, mannitol, mannose, glucose, and the like. The cryoprotective substance may constitute 5 to 30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, at 5 to 30% (e.g., 10%) (w / v). After formulation with the cryoprotective substance, the LNP composition may be frozen at -20°C to -80°C (or lyophilized and cryogenically stored).

[0099] The LNP composition may be administered to the patient in an aqueous buffer solution, or if it is pre-frozen or pre-lyophilized, it is thawed and reconstituted in the aqueous buffer solution at the bedside. In particularly exemplary embodiments, the buffer solution is isotonic and suitable, for example, for intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate-buffered saline (PBS).

[0100] II. RNA The LNP vaccine compositions of this disclosure may include an RNA molecule (e.g., mRNA) encoding the antigen of interest. The RNA molecule of this disclosure may include at least one ribonucleic acid (RNA) containing an ORF encoding the antigen of interest. In certain embodiments, the RNA is messenger RNA (mRNA) containing an ORF encoding the antigen of interest. In certain embodiments, the RNA (e.g., mRNA) is at least one 5'UT R, 3' UTR, poly(A) tail, and / or 5' cap are further included.

[0101] II.A. 5' Cap mRNA 5' caps can confer resistance to nucleases found in most eukaryotic cells and promote translational efficiency. Several types of 5' caps are known, including the 7-methylguanosine cap ("m"). 7Also known as "G" or "CAP-0", it contains guanosine linked to the first transcribed nucleotide via a 5'-5' triphosphate bond.

[0102] 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 by guanylyltransferase to form a 5'5'5 triphosphate bond; and then the 7-nitrogen of guanine is methylated by methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication 2016 / 0032356 and U.S. Patent Application Publication 2018 / 0125989, which are incorporated herein by reference.

[0103] 5'-capping of polynucleotides may be completed simultaneously during an in vitro transcription reaction using the following chemical RNA cap analogues that produce 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). 5'-capping of the modified RNA may be completed post-transcriptionally using a vaccinia virus capping enzyme to produce the cap 0 structure: m7G(5')ppp(5')G. The cap 1 structure may be produced using both a vaccinia virus capping enzyme and a 2'-O methyltransferase to produce m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure may be produced from the cap 1 structure, followed by 2'-O methylation of the 5'-antepenultimate nucleotide using a 2'-O methyltransferase. The cap 3 structure may be produced from the cap 2 structure, followed by 2'-O methylation of the 5'-preantepenultimate nucleotide using a 2'-O methyltransferase.

[0104] In a particular embodiment, the mRNA of the Disclosure includes 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.

[0105] In certain embodiments, the mRNA of this disclosure is [ka] Includes a 5' cap.

[0106] II.B. Untranslated Regions (UTR) In some embodiments, the mRNA of this disclosure includes 5' and / or 3' untranslated regions (UTRs). 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 following the stop codon and continues to the transcription termination signal.

[0107] 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 65 The lengths are 0 nucleotides, approximately 700 nucleotides, approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, approximately 900 nucleotides, approximately 950 nucleotides, approximately 1,000 nucleotides, approximately 1,500 nucleotides, approximately 2,000 nucleotides, approximately 2,500 nucleotides, approximately 3,000 nucleotides, approximately 3,500 nucleotides, approximately 4,000 nucleotides, approximately 4,500 nucleotides, or 5,000 nucleotides.

[0108] In some embodiments, the mRNA disclosed herein may include a 3'UTR containing one or more polyadenylation signals, a protein binding site affecting the stability of the mRNA's position within the cell, or one or more miRNA binding sites. In some embodiments, the 3'UTR may be approximately 50 to 5,000 nucleotides long or longer. In some embodiments, the 3'UTR may be approximately 50 to 1,000 nucleotides long 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 5,000 nucleotides long.

[0109] 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).

[0110] In certain embodiments, the 5' and / or 3'UTR sequences can be derived from stable mRNA (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzyme) to increase mRNA stability. For example, the 5'UTR sequence may include a partial sequence or fragment thereof of the CMV Early 1 (IE1) gene to improve nuclease resistance and / or improve mRNA half-life. It is also intended that the 3' end or untranslated region of the mRNA may include a sequence or fragment thereof encoding human growth hormone (hGH). Generally, these modifications improve mRNA stability and / or pharmacokinetic properties (e.g., half-life) compared to their unmodified counterparts, and include modifications made to improve such mRNA resistance to in vivo nuclease digestion, for example.

[0111] Exemplary 5'UTRs include sequences derived from the CMV earliest 1(IE1) gene (U.S. Patent Application Publication No. 2014 / 0206753 and U.S. Patent Application Publication No. 2015 / 0157565, each of the aforementioned patents incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO: 22) (U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference).

[0112] 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 Publication 2017 / 0029847, U.S. Patent Application Publication 2016 / 0304883, U.S. Patent Application Publication 2016 / 0235864, and U.S. Patent Application Publication 2016 / 0166710, each of the aforementioned patent documents incorporated herein by reference).

[0113] In a particular embodiment, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (see U.S. Patent Application Publication 2017 / 0029847, above).

[0114] In a particular embodiment, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (see U.S. Patent Application Publication 2016 / 0166710, above).

[0115] In a particular embodiment, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (see U.S. Patent Application Publication 2016 / 0166710, above).

[0116] In some embodiments, the intra-sequence ribosome entry site (IRES) is used instead of the 5'UTR.

[0117] In some embodiments, the 5'UTR includes the nucleic acid sequence shown in SEQ ID NO: 19. In some embodiments, the 3'UTR includes the nucleic acid sequence shown in SEQ ID NO: 20. The 5'UTR and 3'UTR are described in more detail in WO2012 / 075040, which is incorporated herein by reference.

[0118] II.C. Polyadenylated 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 provide stability to mRNA and protect it from exonuclease degradation. Poly(A) tails can enhance translation. In some embodiments, poly(A) tails are essentially homopolymers. For example, a poly(A) tail of 100 adenosine nucleotides may essentially have a length of 100 nucleotides. In certain embodiments, a poly(A) tail may be interrupted by at least one nucleotide different from the 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 the adenosine nucleotides, or a sequence of nucleotides). In a particular embodiment, the poly(al)tail includes photograph AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (Sequence ID 23).

[0119] As used herein, “poly(A)tail” typically refers to RNA. However, in the context of this disclosure, the term also refers to the corresponding sequence in a DNA molecule (e.g., “poly(T) sequence”).

[0120] 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.

[0121] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In certain embodiments, the poly(A) tail is obtained by common chemical synthesis methods without transcription from a DNA template. In various embodiments, the poly(A) tail is produced by enzymatic polyadenylation of RNA using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using methods and means described in WO2016 / 174271, for example, using immobilized poly(A) polymerase.

[0122] Nucleic acids may contain poly(A) tails obtained by enzymatic polyadenylation, and the majority of nucleic acid molecules contain approximately 100 (+ / -20) to 500 (+ / -50) or 250 (+ / -20) adenosine nucleotides.

[0123] In some embodiments, the nucleic acid may include a poly(A) tail derived from template DNA, and may further include at least one additional poly(A) tail produced by enzymatic polyadenylation, for example, as described in WO2016 / 091391, the aforementioned patent document is incorporated herein by reference.

[0124] In a particular embodiment, the nucleic acid includes at least one polyadenylation signal.

[0125] In various embodiments, the nucleic acid may contain at least one poly(C) sequence.

[0126] The term "poly(C) sequence," as used herein, is intended to refer to a sequence of cytosine nucleotides up to approximately 200 cytosine nucleotides. In some embodiments, The poly(C) sequence contains approximately 10 to 200 cytosine nucleotides, approximately 10 to 100 cytosine nucleotides, approximately 20 to 70 cytosine nucleotides, approximately 20 to 60 cytosine nucleotides, or approximately 10 to 40 cytosine nucleotides. In some embodiments, the poly(C) sequence contains approximately 30 cytosine nucleotides.

[0127] II.D. Chemical Modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may contain at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications may include skeletal modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) containing, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNAs include, 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), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bro These can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines such as mo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyluracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methylcarbonylmethyl-uracil, 5-methoxyuracil, uracil-5-oxyacetate methyl uracil, uracil-5-oxyacetate(v), 1-methyl-psoidouracil, quosin, β-D-mannosylquosin, phosphoramic acid, phosphorothioate, peptide nucleotides, methylphosphonic acid, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0128] In some embodiments, the disclosed mRNA may contain at least one chemical modification, including but not limited to pseudouridine, N1-methylpsoiduridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-psoiduridine, 2-thio-1-methylpsoiduridine, 2-thio-5-aza-uridine, 2-thio-dihydropsoiduridine, 2-thio-dihydrouridine, 2-thiopsoiduridine, 4-methoxy-2-thiopsoiduridine, 4-methoxypsoiduridine, 4-thio-1-methylpsoiduridine, 4-thiopsoiduridine, 5-aza-uridine, dihydropsoiduridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0129] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpsoiduridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0130] In some embodiments, the chemical modification includes N1-methylpsoiduridine.

[0131] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, and at least 70% of the uracil nucleotides in mRNA. %, at least 80%, at least 85%, at least 90%, at least 95%, or 100% are chemically modified.

[0132] 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.

[0133] The manufacture of such similar products is described, for example, in U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642.

[0134] II.E. mRNA synthesis The mRNA disclosed herein may be synthesized according to a wide variety of methods. For example, mRNA synthesized according to this disclosure can be synthesized by in vitro transcription (IVT). Several methods for in vitro transcription are described, for example, Geall et al., (2013) Semin.Immunol.25(2):152-159; 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 contain DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions may vary depending on the 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. While mRNA provided from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals, can be used in accordance with this disclosure.

[0135] III. Method for producing this LNP vaccine The LNP can be produced by various techniques currently known in the art. For example, multilayer vesicles (MLVs) may be produced according to conventional techniques such as dissolving lipids in a suitable solvent, then evaporating the solvent to leave a thin film on the inside of a vessel, or depositing selected lipids on the inner wall of a suitable container or vessel by spray drying. The MLV may then be formed by adding the aqueous phase to a vortexing vessel. Monolayer liposomes (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilayer vesicles. Furthermore, monolayer liposomes can be formed by detergent removal techniques.

[0136] Various methods are described in US2011 / 0244026, US2016 / 0038432, US2018 / 0153822, US2018 / 0125989, and PCT / US2020 / 043223 (filed July 23, 2020) and can be used to carry out the present invention. One exemplary method, as described in US2016 / 0038432, requires encapsulating mRNA by mixing mRNA with a lipid mixture without first forming lipid nanoparticles from the lipids. Another exemplary method, as described in US2018 / 0153822, requires encapsulating mRNA by mixing mRNA with pre-formed LNPs.

[0137] In some embodiments, a method for producing mRNA-loaded LNPs includes the step of heating one or more solutions to a temperature higher than ambient temperature, wherein one or more solutions are a solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing LNP-encapsulated mRNA. In some embodiments, the method includes the step of heating one or both of the mRNA solution and the pre-formed LNP solution prior to the mixing step. In some embodiments, the method includes heating one or more of the solution containing the pre-formed LNP, the solution containing mRNA, and the solution containing LNP-encapsulated mRNA during the mixing step. In some embodiments, the method includes the step of heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature at which one or more of the solutions are heated is about 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.

[0138] Various methods may 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 encapsulation. 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 encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or buffer 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.

[0139] In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate of at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, or 20× of the mRNA stock solution rate. 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 a flow rate greater than that.

[0140] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10 to 600 ml / min (e.g., 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 in the range of approximately 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 2 The mixture is mixed at a flow rate of 5 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or greater.

[0141] The method of incorporating a desired mRNA into lipid nanoparticles is called "loading." An exemplary method is described by Lasic et al., FEBS Lett. (1992) 312: pp. 255-258. The LNP-incorporated nucleic acid may be located entirely or partially within the internal space of the lipid nanoparticle, within the bilayer 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 completely or substantially contained within the internal space of the lipid nanoparticle.

[0142] Appropriate LNPs can be produced in various sizes. In some embodiments, a reduction in the size of lipid nanoparticles is associated with more efficient mRNA delivery. The selection of an appropriate LNP size may take into account the site of the target cell or tissue and, to some extent, the intended application for which the lipid nanoparticles are produced.

[0143] A wide variety of methods known in the art can be used for size classification of lipid nanoparticle populations. A particularly exemplary method described herein is the zetasizing nano (ZS) (Malvern LNP particle size is measured using Panalytical (Panalytical) analysis. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed in a zetasizing instrument. The z-mean diameter (nm), or cumulant mean, is considered the average size of the LNPs in the sample. Using a zetasizing instrument, the polydispersity index (PDI) can also be measured by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The mean LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles may be repeated alternately with quasi-elastic light scattering (QELS) evaluation to lead to efficient lipid nanoparticle synthesis.

[0144] In some embodiments, the majority of the purified LNPs, i.e., more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70–150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., more than 80% or 90%) of the purified lipid nanoparticles have a size of about 70–150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0145] 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.

[0146] In some embodiments, more than about 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., 55–65 nm), which is particularly suitable for pulmonary delivery by inhalation.

[0147] In some embodiments, the degree of dispersion of LNP in the pharmaceutical composition provided by the present invention, or The molecular size heterogeneity measure (PDI) is less than about 0.5. In some embodiments, 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 the zetasizing instrument described above.

[0148] In some embodiments, more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., more than 80% or 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 90%, or more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 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%).

[0149] 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, a typical LNP of this specification has an N / P ratio of 4.

[0150] In some embodiments, the pharmaceutical composition according to the present invention contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, the pharmaceutical composition contains at least 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.

[0151] In some embodiments, mRNA can be produced by chemical synthesis or by in vitro transcription (IVT) of a DNA template. An exemplary method for producing and purifying mRNA is described in Example 1. In this method, the IVT method uses a cDNA template to produce mRNA transcripts, which are then degraded by DNase. The transcripts are purified by deep filtration and tangent flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is again purified by deep filtration and TFF.

[0152] 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, phosphoric acid, acetic acid, 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, potassium, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0153] An exemplary, non-limiting method for preparing mRNA-LNP compositions is described in Example 1. The method involves mixing a buffered mRNA solution with a lipid solution in ethanol in a controlled and homogeneous manner, wherein the lipid-to-mRNA ratio is maintained throughout the mixing process. In this example, mRNA is prepared using citrate monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is presented in an aqueous buffer containing um. 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 aqueous mRNA solution and the ethanol lipid solution are mixed in a 4:1 volume ratio in a "T" mixer having a nearly "pulseless" pump system. The resulting mixture then undergoes downstream purification and buffer exchange. Buffer exchange may be achieved using a dialysis cassette or a TFF system. The TFF may be used to concentrate and buffer exchange the initial LNPs obtained immediately after formation by the T-mixing method. The diafiltration method is a continuous operation, maintaining a constant volume by adding the appropriate buffer at the same rate as the permeate flow.

[0154] IV. Packaging and Use of mRNA-LNP Vaccines mRNA-LNP vaccines can be packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or nasopharyngeal (e.g., intranasal) administration. The vaccine composition may also be in the form of an immediate formulation, and the LNP composition may be lyophilized and reconstituted with physiological buffer (e.g., PBS) immediately before use. The vaccine composition may be transported and provided in the form of an aqueous solution or a frozen aqueous solution, and can be administered directly to the subject without reconstitution (after thawing if previously frozen).

[0155] Accordingly, this disclosure provides a product such as a kit that provides an mRNA-LNP vaccine in a single container, or provides an mRNA-LNP vaccine in one container and a physiological buffer for reconstitution in another container. The container(s) may contain a single-dose dose or a multi-dose dose. The container(s) may be pre-treated glass vials or ampoules. The product may similarly include instructions for use.

[0156] 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, the deltoid muscle of the upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for use by inhalation and is provided in a pre-filled pump, an endotracheal spray, or an inhaler.

[0157] mRNA-LNP vaccines are administered to those in need in a prophylactically effective 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, be the prevention or reduction of symptoms associated with infection by the pathogen. In some embodiments, multiple doses (e.g., two doses) of the vaccine are injected into those in need to achieve the desired prophylactic effect. The administrations (e.g., prime and booster doses) may be separated by intervals of, for example, 1 week, 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.

[0158] In some embodiments, a single-dose mRNA-LNP vaccine contains 1 to 50 μg of mRNA (e.g., monovalent or polyvalent). For example, a single dose may contain approximately 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, or 15 μg of mRNA for intramuscular (IM) injection. In further embodiments, a polyvalent single-dose LNP vaccine contains multiple (e.g., 2, 3, or 4) types of LNPs for each different antigen, with each type of LNP having, for example, approximately 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, or 15 μg of mRNA.

[0159] In another aspect, the present invention provides a method for immunizing a subject to one or more influenza viruses. Further methods are provided for inducing an immune response against the virus. In some embodiments, the method involves administering an effective amount of the composition described herein to a subject.

[0160] 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 compositions described herein can provide broad cross-protection against different types of influenza viruses. In some embodiments, the compositions provide cross-protection against avian, porcine, seasonal, and / or pandemic influenza viruses. In some embodiments, the compositions provide cross-protection against one or more influenza A, B, or C subtypes. In some embodiments, the compositions 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).

[0161] In some embodiments, the methods of the present invention can induce an improved immune response against one or more seasonal influenza strains. Exemplary seasonal strains include A / Puerto Rico / 8 / 1934, A / Fort Monmouth / 1 / 1947, A / Chile / 1 / 1983, A / Texas / 36 / 1991, A / Singapore / 6 / 1986, A / Beijing / 32 / 1992, A / New Caledonia / 20 / 1999, and A / Solomon. Islands / 03 / 2006, A / Brisbane / 59 / 2007, A(H3N2) virus antigenically similar to cell-transmitting 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(H1 N1) virus, A / California / 7 / 2009(H1N1)pdm09 virus, A / Cambodia / e0826360 / 2020(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 A / Hong Kong / 2671 / 2019(H3N2) virus, A / Hong Kong / 45 / 2019(H3N2) virus, A / Hong Kong / 4801 / 2014(H3N2) virus, A / Kansas / 14 / 2017(H3N2) virus, A / Michigan / 45 / 2015(H1N1)pdm09 virus, A / Moscow / 10 / 99(H3N2) virus, A / New Caledonia / 20 / 99(H1N1) virus, A / Perth / 16 / 2009(H3N2) virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus, A / SolomonIslands / 3 / 2006(H1N1) virus, A / South Australia / 34 / 2019(H3N2) virus, A / Switzerland / 8060 / 2017(H3N2) virus, A / Switzerland / 9715293 / 2013(H3N2) virus, A / Sydney / 5 / 97(H3N2) virus, A / Texas / 50 / 2012(H3N2) virus, A / Victoria / 2570 / 2019(H1N1)pdm09 virus, A / Victoria / 2570 / 2019(H1N1)pdm09 virus, A / Victoria / 361 / 2011(H3N 2) Viruses such as A / Wellington / 1 / 2004(H3N2), A / Wisconsin / 588 / 2019(H1N1)pdm09, A / Wisconsin / 588 / 2019(H1N1)pdm09, A / Wisconsin / 67 / 2005(H3N2), B / Beijing / 184 / 93, B / Brisbane / 60 / 2008, B / Colorado / 06 / 2017 (B / Victoria / 2 / 87 lineage), B / Florida / 4 / 2006, B / Hong This includes, without limitation, the following viruses: Kong / 330 / 2001, B / Malaysia / 2506 / 2004, B / Massachusetts / 2 / 2012, B / Phuket / 3073 / 2013 (B / Yamagata lineage), B / Phuket / 3073 / 2013, B / Phuket / 3073 / 2013 (B / Yamagata / 16 / 88 lineage), B / Shangdong / 7 / 97, ​​B / Shanghai / 361 / 2002, B / Sichuan / 379 / 99, B / Washington / 02 / 2019 (B / Victoria lineage), B / Washington / 02 / 2019 (B / Victoria lineage), and B / Wisconsin / 1 / 2010. In some embodiments, the methods of the present invention can induce an improved immune response against 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. Pandemic subtypes include, in particular, the 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 against one or more swine influenza strains.Exemplary porcine strains include, but are not limited to, the A / New Jersey / 1976 isolate and the A / California / 07 / 2009 isolate. 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 influenza pandemic, seasonal, avian, and / or porcine strains are known in the art.

[0162] In some embodiments, the present invention provides a method for preventing or treating influenza infection by administering a composition of the present invention 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 to have influenza infection if he or she exhibits one or more symptoms generally associated with influenza infection. In some embodiments, the subject is known or suspected to have been exposed to the influenza virus. In some embodiments, the subject is considered susceptible to influenza infection if he or she is known or suspected to have been exposed to the influenza virus. In some embodiments, the subject is known or suspected to have been exposed to the influenza virus if he or she has been in contact with another individual who is known or suspected to be infected with the influenza virus and / or if he or she is in or has been in a location where an influenza outbreak is known or suspected to be occurring.

[0163] In various embodiments, the compositions described herein may be administered prior to or after the onset of one or more symptoms of influenza infection. In some embodiments, the compositions are administered as prophylactic agents. In such embodiments, the methods of the present invention are effective in preventing or protecting a subject from influenza virus infection. In some embodiments, the compositions of the present invention are intended to be components of seasonal and / or pandemic influenza vaccines or to provide sustained (multiseason) protection. It is used as part of a prescribed influenza vaccination regimen. In some embodiments, the compositions of the present invention are used to treat the symptoms of influenza infection.

[0164] In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is livestock or pet (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., chickens, ducks, geese, and / or turkeys).

[0165] In some embodiments, the subject is human. In certain embodiments, the subject is adult, adolescent, or infant. In some embodiments, the human subject is younger than 6 months of age. In some embodiments, the human subject is 6 months of age or older, 6 months to 35 months of age, 36 months to 8 years of age, or 9 years of age or older. In some embodiments, the human subject is 60 years of age or older, or elderly, such as 55 years of age or older, or 65 years of age or older. Intrauterine administration and / or treatment methods of the composition are also intended herein.

[0166] Unless otherwise defined herein, scientific and technical terms used in connection with this specification shall have the same meaning as those commonly 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 execution or testing of the present invention. In case of any conflict, this specification, including its definitions, shall prevail. 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 pharmaceutical chemistry, and 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 as commonly performed in the art or as described herein, in accordance with the manufacturer's specifications. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. Throughout this specification and its embodiments, the words “have” and variations such as “comprise” or “has,” “having,” “comprises,” or “comprising” are understood to mean including the stated integer or group of integers, 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. Although several documents are cited herein, this citation does not constitute an acknowledgment that any of these documents form part of the universal and general knowledge of the art. As used herein, the terms “approximately” or “about,” applied to one or more values ​​of interest, mean a value that is similar to the stated reference value. In certain embodiments, unless otherwise stated or evident from the context, this term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value.

[0167] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. [Examples]

[0168] Optimization of LNP formulations This example describes a study in which a series of LNP formulations for mRNA vaccines were prepared from a combinatorial library of various components. It also describes a rationally designed novel cationic lipid. The following were synthesized. In total, more than 150 lipids and more than 430 formulations were tested. Human erythropoietin (hEPO) mRNA was used as the test mRNA. In the major formulations listed below, the mRNA is formulated into LNPs using a combination of cationic lipids and three other lipids: helper lipids; cholesterol lipids; and pegylated lipids, in various permutations.

[0169] The LNP formulation consists of four lipid components: ionizable lipids, the helper lipid DOPE, cholesterol, and the pegylated lipid DMG-PEG-2K. The molar fraction of the pegylated lipid was kept constant at 1.5%, and the ionizable lipids and different helper lipids and their molar ratios were evaluated to confirm the optimal ratio based on hEPO screening studies.

[0170] Citrate buffer (1 mM citrate, 150 mM NaCl, pH 4.5) was used in the preparation of the LNP formulation. The mRNA solution added to the citrate buffer was mixed with lipids in an ethanol solution during the formulation process. The pH and concentration of the buffer were selected to achieve a high rate of mRNA encapsulation in the LNP formulation.

[0171] The LNP formulation method involved mixing a lipid ethanol solution and an mRNA citrate solution in a "T" mixer using a pump system. The resulting solution was then subjected to buffer exchange using a TFF / dialysis tube. The concentration of the final formulation in 10% (w / v) trehalose was adjusted based on the administration requirements.

[0172] In vivo expression of hEPO protein in mice was used as a surrogate to measure the efficacy of LNPs that deliver mRNA in vivo. In this study, a single dose (0.1 μg) of hEPO mRNA formulated in LNPs derived from various combinations of components was injected intramuscularly (IM) into mice. Serum collected at 6 and 24 hours post-administration was tested for hEPO levels using ELISA. The industry benchmark MC3 formulation was used as a reference for calculating the multiplier of hEPO expression increase (Angew, Chem Int Ed. (2012) 51:85 pp. 29-33).

[0173] The level of hEPO expression observed for each LNP formulation indicated the formulation's ability to deliver mRNA into cells. The first formulation contained 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; helper lipid), DMG-PEG2000, and cholesterol in a molar ratio of cationic lipid:DMG-PEG2000:cholesterol:DOPE of 40:1.5:28.5:30. These formulations were found to have more robust efficacy compared to MC3 formulations.

[0174] Further formulations were tested. Optimized formulation lipid A LNPs and lipid B LNPs are shown in Table 1. The mRNA in these formulations can be modified or unmodified and may encode virus-derived antigens such as influenza or SARS-CoV-2.

[0175] [Table 1]

[0176] Table 1 indicates that the final dose for human vaccines is considered to be a dilution of the final bulk product in phosphate-buffered saline (PBS) based on the intended single human dose. The WFI amount is calculated based on the nominal final formulation. The trehalose content in the formulation is equivalent to 10% (100 mg / mL) trehalose dihydrate and is converted to the anhydrous component using the ratio of the molecular weights of anhydrous trehalose to trehalose dihydrate.

[0177] The molar ratios of lipid components in the two optimized formulations, lipid A and lipid B LNP formulations, are shown in Table 2 (CL: cationic lipid).

[0178] [Table 2]

[0179] As shown in Table 3 and Figure 1A, the multiplier increase in hEPO expression in lipids A and B compared to MC3 indicates that these LNPs are superior to MC3 in mRNA delivery. In the table below, "P2" refers to PEG2000; "Times MC3" refers to the multiplier of increase compared to MC3; and "Std Dev" refers to the standard deviation.

[0180] [Table 3-1] [Table 3-2]

[0181] Figure 1B shows hEPO expression in mice and non-human primates (NHPs) using LNP lipids A and B. A single dose of hEPO mRNA formulated with lipid A or B (0.1 μg in mice and 10 μg in NHPs) was administered intramuscularly. Serum hEPO levels were quantified at 6, 24, 48, and 72 hours post-administration using ELISA. The data demonstrate long-term in vivo hEPO protein expression beyond 4 days in mice and NHPs.

[0182] One of the key process parameters identified during optimization is the flow rate during the initial mixing step. Formulations with different final LNP sizes (ranging from 108 to 177 nm) are manufactured by varying these flow rates during mixing, allowing for additional control over the method and product attributes. As the flow rate increases, the particle size decreases accordingly. When the flow rate reached 375 ml / min and LNPs with an average size of 108 nM were produced, the efficacy increased significantly. The effect of LNP size on efficacy was noted as a measure of the increase in hEPO expression relative to MC3, as shown in Table 4.

[0183] [Table 4]

[0184] The above screening data indicates that the helper lipid DOPE was effective in promoting protein expression. The data also led to the determination of promising molar compositions of four lipids (OF-02 or cKK-E10:DMG-PEG-2K:cholesterol:DOPE = 40:1.5:28.5:30). LNP formulations in 10% trehalose were characterized for all parameters, including particle size, PDI, mRNA encapsulation, and mRNA integrity. All tested batches exhibited the desired characteristics and freeze / thaw cycle stability. Long-term stability of formulations in 10% (w / v) trehalose at -80°C was evaluated. Lipid A and lipid B formulations were found to be highly stable. [Examples]

[0185] Influenza HIN1 LNP vaccine preparation The emergence of a novel influenza virus in a human population can lead to an influenza pandemic. Such pandemics remain a significant threat to public health and require vigilance and preparedness with protective measures to be used in the event of prolonged human-to-human transmission of the virus. In the experiments described in this embodiment, the efficacy of mRNA vaccines prepared with lipid A and lipid B LNP formulations was evaluated using hemagglutinin (HA) derived from the highly pathogenic HIN1 strain A / California / 7 / 2009 (CA09), which caused the 2009 influenza pandemic, as a prototype antigen.

[0186] HA mRNA was prepared as described above. Citrate buffer (1 mM citrate, 150 mM NaCl, pH 4.5) was used in the preparation of the LNP composition. The citrate buffer containing mRNA was mixed with lipids in an ethanol solution during the formulation process. The pH and concentration of the buffer were selected to achieve a high encapsulation rate of mRNA in the LNP formulation. The two solutions (mRNA in citrate buffer and lipids in ethanol solution) were mixed in a "T" mixer using a pump system to obtain a homogeneous pulseless flow, and the lipids and mRNA were mixed in a constant ratio throughout the method. This was crucial to achieving a homogeneous formulation with the desired size and a low PDI, which is an indicator of a more homogeneous size distribution. This method resulted in high mRNA encapsulation, which is crucial for achieving high potency. The solution thus obtained was then subjected to buffer exchange using TFF / dialysis tubing.

[0187] In mouse studies, the efficacy of lipid A and lipid B CA09 HA formulations was evaluated by cross-referencing with MC3 LNP formulations and recombinant HA (rHA). CA09(H1)HA mRNA (0.4 μg) formulated with different cationic lipids was injected intramuscularly into Balb / C mice (n=8) on day 0 (D0) and day 28 (D28). Vaccine immunogenicity was indicated by HA inhibitory (HAI) titer, as shown in Figure 2A. The data show that immunization with either lipid A or lipid B on day 0 (D0) and day 28 (D28) induced high HAI titers, enabling complete protection of the animals from homologous viral challenge (Belgium 09 H1N1 virus) (Figure 2B). During the 14-day post-challenge observation period, no obvious signs of pathological conditions (weight loss) were observed in the lipid A and lipid B treatment groups, while a small number of animals in the recombinant protein control group exhibited pathological conditions (Figure 2B).

[0188] Similarly, mRNA encoding neuraminidase (NA) from the Mich15 influenza strain (Mich15 N1) was formulated using lipid A, and its efficacy was evaluated. Two doses (0.4 or 0.016 μg) of NA mRNA formulated with lipid A were injected intramuscularly into Balb / c mice (n=8). The control group (n=8) was injected with 0.6 μg or a dilution of hEPO mRNA. Half of the mice received only one injection (one dose) on day 0 of the study, while the other half received two injections (two doses) on day 0 and day 28 of the study. The data show that this N1 lipid A formulation induced a robust immune response, as indicated by NA inhibitory (NAI) titers (Figure 3A). The data further show that mice treated with one or two doses of the vaccine were protected from lethal viral challenge by Belgium 09 H1N1 (Figure 3B). The level of protection correlated with the NAI titer of the vaccine-treated group versus the negative control group (hEPO and diluent).

[0189] CA09 H1 mRNA formulated using this LNP was also tested in an NHP model. mRNA (10 μg) was formulated with lipids A and B and injected intramuscularly into cynopasu monkeys (n=6) on days 0 and 28 of the study. Detectable HAI priming up to day 14 and a significant boost in HAI titer up to day 28 were observed for all LNPs (Figure 4, right panel). ELISA data also showed significant above-baseline priming by day 14 for all doses tested, with a robust boost detected two weeks after the boost (Figure 4, left panel). The results demonstrate that this H1 mRNA formulation induced a robust immune response, as indicated by HAI and endpoint ELISA titers. [Examples]

[0190] Influenza H3N2 LNP vaccine preparation This example describes experiments evaluating the efficacy of mRNA-LNP vaccine formulations against influenza strain Sing16(H3N2). One of the mRNAs used in these experiments is MRT1400. MRT1400 is a biosynthetic codon-optimized HA-H3 (influenza virus hemagglutinin, H3 subtype) messenger RNA (CO-HA-H3 mRNA) produced by in vitro transcription.

[0191] The protein sequences for influenza virus hemagglutinin, H3 subtype, are shown below. MKTIIALSYI LCLVFAQKIP GNDNSTATLC LGHHAVPNGT IVKTITNDRI EVTNATELVQ NSSIGEICDS PHQILDGENC TLIDALLGDP QCDGFQNKKW DLFVERSKAY SNCYPYDVPD YASLRSLVAS SGTLEFKNES FNWTGVTQNG TSSACIRGSS SSFFSRLNWL THLNYTYPAL NVTMPNKEQF DKLYIWGVHH PGTDKDQIFL YAQSSGRIT V STKRSQQAVI PNIGSRPRIR DIPSRISIYW TIVKPGDILL INSTGNLIAP RGYFKIRSGK SSIMRSDAPI GKCKSECITP NGSIPNDKPF QNVNRITYGA CPRYVKHSTL KLATGMRNVP EKQTRGIFGA IAGFIENGWE GMVDGWYGFR HQNSEGRGQA ADLKSTQAAI DQINGKLNRL IGKTNEKFHQ IEKEFSEVEG RVQDLEKYVE DTKIDLWSYN AELLVALENQ HTIDLTDSEM NKLFEKTKKQ LRENAEDMGN GCFKIYHKCD NACIESIRNE TYDHNVYRDE ALNNRFQIKG VELKSGYKDW ILWISFAISC FLLCVALLGF IMWACQKGNI RCNICI*(Sequence ID 1)

[0192] The coding sequence for this protein was codon-optimized. The codon-optimized sequence coding the protein is shown in Figure 5A (SEQ ID NO: 2), and the wild-type sequence is shown as SEQ ID NO: 3. The mRNA structure and sequence are shown in Figures 5B and 5C, respectively. As shown in the figures, the HA-H3 mRNA coding sequence has 140 and 100 nucleotides of 5' and 3' untranslated regions (UTRs) adjacent to each other. The biosynthetic HA-H3 mRNA has 7-methylguanosine (m) linked to the first nucleoside of the 5'UTR via an inverted 5'-5' triphosphate crosslink. 7 It also contains a 5' cap structure consisting of residue G), and this first nucleoside is itself modified by 2'-O-ribose methylation. The 5' cap is essential for the initiation of translation by ribosomes. The entire linear structure terminates at the 3' end with a tract (poly-A) of approximately 100-500 adenosine nucleosides. The poly-A region is thought to provide stability to the mRNA and enhance translation. All of these structural elements are naturally occurring components used to promote efficient translation of HA-H3 mRNA.

[0193] DNA plasmids were constructed to produce codon-optimized mRNA sequences by in vitro transcription. The in vitro transcription (IVT) reaction was performed using RNA polymerase. The reaction mixture was allowed to precipitate. The precipitated RNA samples were loaded onto individual deep filtration cassettes, washed with 80% ethanol, and redissolved in recirculated water. A second aliquot of water was dispensed in a manner similar to the first step. This step was repeated once more. The pooled eluates were subjected to ultrafiltration / diafiltration using 50kD hollow fiber TFF cassettes. Each IVT TFF pool was then diluted in preparation for the cap-tail reaction. The cap-tail reaction was allowed to precipitate, and the RNA derived from the reaction was purified and collected as described above. The filtered mRNA was stored at -20°C until use.

[0194] In these experiments, mRNA encoding the Sing16 NA(N2) or Sing16 HA(H3; MRT1400 mRNA) antigen was formulated using lipid A or lipid B and injected intramuscularly into Balb / c mice (n=8) at a dose of 0.4 μg of mRNA per day on days 0 and 28. For comparison, 1 μg of recombinant Sing16 H3 or Sing16 N2 protein was injected intramuscularly into Balb / c mice (n=8) together with an oil-in-water emulsion adjuvant (AF03). Immune responses were measured by NAI and HAI assays.

[0195] The data show that animals immunized with NA(N2) mRNA demonstrated detectable NAI priming by day 14 and a significant boost in NAI titers by day 28 (Figure 6, right panel). The data also show that HA Sing16 lipid A and lipid B formulations induced a robust HAI response after the boost at day 28 (Figure 6, left panel).

[0196] Similarly, the Sing16 HA mRNA lipid A and lipid B vaccines were evaluated in non-human primates (NHP) cynopauses (n=6). Lipid A or lipid B The HA Sing16 mRNA (50 μg) formulation was administered to monkeys via intramuscular injection. The first injection was given on study day 0, and the second injection was given on study day 28. The data show that the vaccine induced a robust immune response boosted on day 28 (Figure 7A).

[0197] Furthermore, four dose levels of HA Sing16 mRNA formulated with lipid A (i.e., MRT5400 vaccine), 15, 45, 135, and 250 μg, were evaluated by NHP. The first immunization was administered on study day 0, and the second immunization on study day 28. All NHPs showed IgG binding and HAI titers for all doses tested, and there were no differences in immune response among the various doses tested two weeks after the second injection on day 42 (Figures 7B and 7C).

[0198] The Sing16 HA mRNA lipid A vaccine was also evaluated for T cell response in NHP after a second vaccination. Peripheral blood mononuclear cells (PBMCs) were collected on day 42 and incubated overnight with a peptide pool representing either Sing16 H3 recombinant protein or the total HA open reading frame. Restimulation-induced cytokines were evaluated by the ELISPOT assay. The frequency of PBMCs secreting IFN-γ, Th1 cytokines (Figure 8A), or IL-13, Th2 cytokines (Figure 8B) was calculated as spot-forming cells (SFCs) per million PBMCs. The majority of animals in the three dose levels tested (250 μg, 135 μg, and 45 μg) showed the presence of high-frequency IFN-γ secreting cells, exceeding 100 SFCs per million PBMCs (Figure 8A). No dose response was observed, as animals in the low and high dose levels showed comparable frequencies of IFN-γ secreting cells. In contrast, the presence of IL-13 cytokine-secreting cells was not detected in any of the tested groups at any dose level (Figure 8B). These data provide clear evidence of the absence of Th1-biased and Th2-biased cell responses to HA antigens following vaccination in NHP. [Examples]

[0199] Influenza LNP vaccine preparations containing modified mRNA This example describes an experiment comparing the efficacy of vaccines containing unmodified (unmodified non-replicated or "UNR") and modified (modified non-replicated or "MNR") mRNA. UNR CA09 HA mRNA and MNR CA09 HA mRNA were prepared by in vitro transcription. In MNR, all uridine molecules were replaced with pseudouridine.

[0200] Five different doses (0.016, 0.08, 0.4, 2, and 10 μg) of CA09 HA mRNA (modified or unmodified) formulated with lipid A were injected into Balb / c mice (n=15) via the intramuscular route. The data showed that the LNP formulation increased the stability and delivery efficiency of naked mRNA (UNR). This was because the potency between UNR and MNR mRNA was comparable, as indicated by HAI titer (Figure 9A). ELISA data for Balb / c mice also showed significant priming above the baseline by day 14 for all doses tested (both UNR and MNR mRNA), and a robust boost was detected two weeks after the boost. The data also show that UNR and MNR mRNA were comparable in inducing ELISA titer (Figure 9B).

[0201] In conclusion, this dose titration study showed that unmodified and modified CA09 HA mRNA formulated with lipid A induced statistically indistinguishable immune responses in Balb / c mice, as indicated by HAI or endpoint ELISA assays. This was demonstrated. Balb / c mice immunized with four higher doses of UNR and MNR mRNA showed detectable HAI priming by day 14 and a significant boost in HAI titer by day 42 for all doses. These day 14 priming titers represent both dose-effectiveness and dose-saving potential, producing detectable titers exceeding a 125-fold range. Second injection titers within the same dose range confirm the robustness of the immune response to this mRNA-LNP formulation. Similar results were observed in non-human primates. [Examples]

[0202] Polyvalent influenza vaccine LNP preparation This example describes a study using a lipid A-based LNP vaccine containing mRNA encoding CA09 HA (described in Example 2) and mRNA encoding Sing16 HA (described in Example 3).

[0203] More specifically, CA09 HA mRNA and Sing16 HA mRNA co-encapsulated in lipid A were evaluated in Balb / c mice (n=8). mRNA-LNP was administered either as two co-encapsulated mRNAs or separately as single-encapsulated mRNAs. In both approaches, a total of 0.4 μg of the LNP preparation was injected into the mice by intramuscular injection. The first injection was given on study day 0, and the second injection was given on study day 28. The data indicate that the vaccine induced a robust immunofunctional response. There appeared to be no difference between the two administration approaches. These data suggest that co-encapsulation did not cause any interference or disruption between the two mRNAs. [Examples]

[0204] Further research on polyvalent influenza vaccine LNP formulations A panel of unmodified mRNAs encoding reporter antigens of CA09 HA, Sing16 HA, Sing16 NA, Mich15 NA, A / Perth / 16 / 2009 influenza virus (Perth09 NA), as well as firefly luciferase (FF) and hEPO was prepared. Next, LNP formulations for HA and NA mRNA-LNP formulations were tested for in vitro expression, immune responses in animals, and efficacy in preclinical models. In the studies in this example, all LNP formulations were lipid A formulations.

[0205] Materials and Methods mRNA-LNP Formulations mRNA transcripts encoding hEPO, FF, CA09 HA, Sing16 HA, Mich15 NA, and Sing16 NA were synthesized by in vitro transcription using RNA polymerase with plasmid DNA templates encoding the desired genes using unmodified nucleotides. The purified mRNA precursors thus obtained were further reacted by enzymatic addition of a 5' cap structure (cap 1) and a roughly 200 nucleotide long 3' poly(A) tail determined and purified by gel electrophoresis. All mRNA formulations were analyzed for purity, integrity, and percentage of cap 1 prior to storage at -20°C. The manufacture of mRNA / lipid nanoparticle (LNP) formulations was described above. Briefly, an ethanol solution of a mixture of lipids at a fixed lipid to mRNA ratio (ionizable lipid, phosphatidylethanolamine, cholesterol and polyethylene glycol-lipid) was combined with an aqueous buffer solution of the target mRNA at acidic pH under controlled conditions to obtain a homogeneous suspension of LNPs. The resulting nanoparticle suspension was diluted to the final concentration by ultrafiltration and diafiltration in a suitable diluent system, filtered, and cryopreserved at -80°C until use. mRNA-LNP formulations were characterized for size by dynamic light scattering, percentage encapsulation, and diluted to 1 mg / mL with the appropriate buffer for further use and stored at - 80°C. hEPO-LNP and FF-LNP were utilized to examine the levels of target protein expression in vivo.

[0206] Visualization of S-protein expressed in HeLa cells Immunocytochemistry-immunofluorescence analysis of influenza NA and HA proteins was performed in HeLa cells transfected with bivalent H3N2 (Sing16 HA and Perth09 NA) mRNA LNP) using the previously described method (Kalnin et al., npj Vaccines (2021) 6:61). Cells were fixed in 4% paraformaldehyde and subjected to antibody staining for HA (GeneTex GTX40258), NA, and the ER marker Calnexin (Abcam ab22595). Images were taken on a confocal microscope and subsequently subjected to image analysis for quantification of HA and NA co-localization to the ER, mean signal intensity, and percentage of cell area.

[0207] Flow cytometry Human skeletal muscle cells (HskMCs, Lonza) were cultured at 37 °C, 5% CO2 in M199 (Life Technologies) supplemented with GlutaMAX (Life Technologies), streptomycin, penicillin (Gibco), and 20% heat-inactivated FBS (VWR). Cells were harvested by trypsinization, washed with PBS, and 10 6 mRNA per cell Electroporation was performed on Nucleofector 2b (Lonza) using a human primary muscle cell transfection kit with 12 mg of the drug. After 24 hours, harvested cells were fixed and permeabilized with Cytofix® / Perm (BD), then stained with CA09 HA (Immune Tech), Sing16 HA (30-2F11-F7-A5, GeneTex), Mich15 NA (6G6, Immune Tech), and Sing16 NA (40017-RP01, Sino Biologicals) specific antibodies, followed by staining with PE conjugate goat anti-mouse IgG secondary antibody (Southern Biotech) or AF647 conjugate goat anti-rabbit IgG (Life Technologies). Next, antibody-labeled cells were obtained using Fortessa (BD), and the expression of each protein was analyzed using FlowJo® (TreeStar).

[0208] Cryogenic transmission electron microscope Prior to LNP sample application, the grid was plasma-cleaned using a PELCO easiGlow® system, and a Vitrobot Mark IV system (ThermoFisher) with the chamber maintained at 100% humidity and 18°C ​​was used for plunge freezing. 3.0 μl droplets of LNP sample were dispensed onto a 300-mesh R2 / 1 QUANTIFOIL® grid with carbon film and gold bars. The grid was blotted for 4 seconds, held in place for 10 seconds, then immediately plunge-frozen in liquid ethane for storage and transferred to a Krios microscope. Exposures were collected using a Titan Krios transmission electron microscope (ThermoFisher) equipped with a BioQuantum energy filter and a K3 direct electron detector (Gatan) operating in counting mode. The calibrated physical pixel size at the detector was 1.38 Å, corresponding to a magnification of 64,000. A total of 3,141 69-frame movie exposures were acquired with a defocus of -0.5 to -1.7 μm and a dose of 1.045 e / Å2 per frame. For each movie exposure, patch-based motion correction, super-resolution pixel binning, and frame weighting were performed using RELION-3.1.34. More than 700 candidate particle coordinates were extracted from the corrected images. Subsequent data analysis was performed using MATLAB R2019a with the image processing toolbox.

[0209] Immunization of mice and NHP for expression studies Four crab-eating macaws (NHP) (male and female) and 4-8 male BALB / c macaques The mice were intramuscularly administered either 10 μg (NHP) or 1, 0.5, 0.1, and 0.05 μg (mouse) along with hEPO-LNP manufactured in the same ratio intended for use with HA / NA mRNA-LNP formulations. Blood samples were collected before administration and at 6, 24, 48, 72, and 96 hours post-administration, and serum hEPO expression was monitored according to the manufacturer's protocol using ELISA with R and D systems, Quantikine® IVD® ELISA, and human erythropoietin immunoassay kits, reported as final values ​​of mIU / ml and ng / ml. Briefly, microplate wells pre-coated with EPO-specific mouse monoclonal antibody were incubated with specimen or standard. After removing excess specimen or standard, the wells were incubated with rabbit anti-EPO polyclonal antibody conjugated to horseradish peroxidase. During the second incubation, the antibody-enzyme conjugate bound to the immobilized EPO. Excess conjugate was removed by washing. A dye source was added to the well, and the dye source was oxidized by an enzymatic reaction to form a blue complex. The reaction was stopped by adding acid, which changed the color from blue to yellow. The amount of color produced was directly proportional to the amount of conjugate bound to the EPO antibody complex, and the amount of the EPO antibody complex was in turn directly proportional to the amount of EPO in the sample or standard. The absorbance of this complex was measured, and a standard curve was created by plotting the absorbance against the concentration of the EPO standard. The EPO concentration of an unknown sample was determined by comparing the optical density of the sample with the standard curve. The standard used in this assay was recombinant hEPO, urine-derived human erythropoietin, calibrated against the second international reference (67 / 343).

[0210] Immunization of mice and NHP for immunogenicity studies Groups of Balb / c mice (Mus musculus) were immunized in the quadriceps via the IM pathway under isoflurane anesthesia with a dose of 0.05 mL of a designated vaccine formulation or diluent, on one hind limb on day 0 and on the opposite limb on day 28. Mice that lost more than 20% of their initial body weight and showed significant clinical signs were euthanized prior to the end of the study, after a veterinary assessment of the animals' health.

[0211] Naive male and female crab-eating macaws (Macaca fascicularis) originating from Mauritius were selected for the study. At the start of the study, the animals weighed >2 kg and were >2 years old. The animals selected for the study underwent extensive physical examination prior to assignment to the study. Pre-assignment health assessment included field veterinary examination and collection of blood samples for CBC analysis applicable according to NIRC SOP. Animals were generally housed in pairs and acclimatized for at least 3 days prior to the start of the study. Groups consisted of a maximum of 6 animals per treatment group. All animals were immunized on study day 0, targeting the deltoid muscle, with 0.5 ml of the respective vaccine formulation or dilution, on one forelimb of each animal via the IM route, under sedation with ketamine HCl (10 mg / kg, IM) or terazole (4-8 mg / kg, IM). A second immunization was administered to the opposite limb of the animal 28 days after the first immunization.

[0212] Immunization of mice and NHP for challenge research Mice were inoculated with the challenge strain approximately 9–12 weeks after their last immunization. The stock virus vials were thawed and diluted to the appropriate concentration in ice-cold sterile PBS. All mice were 4LD 50 Equivalent to 105.54 TCID in PBS 50The challenge was performed with a total volume of 50 μl containing the Belgium 09 virus. The virus challenge was conducted inside a safety cabinet in an expanded ABSL2 laboratory. Mice were first anesthetized by IP injection of ketamine / xylazine solution (ketamine 50 mg / kg and xylazine 5 mg / kg), and then challenged with IN (droplets into both nostrils; 25 μl per nasal cavity) using a micropipette with a total volume of 50 μl of influenza virus. Following the procedure, mice were placed in a supine position and observed until they recovered from anesthesia. Daily weight measurements were taken immediately after the H1N1 challenge. Any individual animal that showed even one instance of >20% weight loss was euthanized. Weight measurements were recorded daily after the challenge until euthanasia in the online database, Pristima® (Version 7.5.0 Build 8), or on a study-specific working sheet.

[0213] Blood collection In mice, blood was collected from all animals under sedation by submandibular or orbital venous plexus hemorrhage (pre-mortem hemorrhage, approximately 200 μl before study and on study days 14, 28, and 42) and cardiac puncture (peripheral hemorrhage, day 56). Mice were bled before study to obtain baseline pre-immunized serum samples for pre-screening. Serum processing and blood samples were collected in SST tubes and allowed to coagulate at room temperature for 30 minutes to 1 hour. The samples were then centrifuged at 1000–1300 g for 5–10 minutes with the brake off. Serum was collected using a P200 pipette, divided into two 0.5 ml cryovials, and stored at -20°C. All bleeding was documented in the sample collection and processing log, indicating the time of sample collection and the technician who performed the procedure. A portion of the serum samples were evaluated for antibody titers in HAI or ELLA and ELISA assays.

[0214] NHPs were anesthetized by intramuscular administration of ketamine 10 mg / kg / acepromazine 1 mg / kg and then bled for serum separation (days 4, 2, 7, 14, 28, 30, 35, 42, 56, 90, and 180). The amount of blood collected did not exceed established guidelines regarding body weight percentage and animal health status. Blood was collected from anesthetized NHPs using femoral vein puncture with a Vacutainer 21 ga × 1” blood collection needle or Abbott Butterfly 23 ga × 3 / 4” tubing attached to a BD Vacutainer® SST® gel tube. Serum was separated by rotating the tube at a rate of 1200 × g for 10 minutes at room temperature. The serum was then divided into labeled cryovials (1 ml / vial) and stored at ≤-20°C. Portions of serum samples were evaluated for antibody titer in HAI or ELLA and ELISA assays. In PBMCs, NHP was used to induce pre-bleeding before vaccination and again approximately 42–63 days after the first injection. For this purpose, blood was collected in BD Vacutainer® tubing containing heparin anticoagulant. Briefly, anticoagulant blood samples were diluted in PBS and subjected to gradient centrifugation at 400 × g for 30 minutes using Histopaque® separator (Sigma). Opaque interfaces containing mononuclear cells were then collected and washed three times with PBS using low-speed (250 × g) centrifugation, with the platelet count reduced in the final centrifugation. Live versus dead PBMCs were counted using a Nexcelom Cellometer K2. PBMCs were cryopreserved in FBS with 10% DMSO using a Mr.Frosty® freezing box. The box was immediately placed in a -80°C refrigerator for 24 hours and then transferred to a liquid nitrogen tank for storage.

[0215] ELISA Antibody ELISA was performed using recombinantly produced Sing16 NA protein, Sing16 HA protein, or CA09 HA protein. Proteins were captured at a concentration of 2 μg / ml in carbonate-bicarbonate buffer on a 96-well high-binding polystyrene plate. The plate was collected and incubated overnight (16 ± 4 hours) at 2–8°C. After overnight incubation, the antigen-coated plate was washed five times with wash buffer (PBS, 0.5% Tween20) and blocked with blocking solution (PBS, 10% BSA) for 60 ± 30 minutes at room temperature. The test sample, naive control, and reference sample were diluted in sample diluent (PBS, 10% BSA, 0.5% Tween20), added to the wells in two ways, and then incubated at room temperature for 90 minutes. The plate was washed five times with wash buffer. Mouse serum was treated with goat anti-mouse HRP at a dilution of 1:10,000, and NHP serum was treated with goat anti-monkey HRP at a dilution of 1:10,000. The plates were then incubated at room temperature for 30 minutes, and excess HRP-IgG was washed off with wash buffer. Sure-Blue TMB substrate was added to each plate, and the reaction was stopped with TMB stop solution after approximately 10 minutes. The plates were then read at 450 nm using a Thermo Labsystems Multiskan™ spectrophotometer. Anti-antigen (HA or NA) specific antibody titers were expressed as the reciprocal of the highest serum dilution with an absorbance value of >0.3.

[0216] HAI assay HAI assays were performed using Sing16 H3N2 and CA09 H1N1 virus stocks (BIOQUAL, Inc.). Serum was treated with receptor-destroying enzyme (RDE) by diluting 1 part serum with 3 parts 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 PBS to a final dilution of 1 / 10. HAI assays were performed in V-bottom 96-well plates using 4 hemagglutination units (HAU) of virus and 0.5% turkey RBCs. Reference serum for each strain was included as a positive control on all assay plates. Each plate also included a back titration to confirm the antigen dose (4 HAU / 25 μl) and a negative control sample (PBS or naive control serum). HAI titer was determined as the highest serum dilution that resulted in complete inhibition of hemagglutination. The results were valid only for plates with appropriate back titration results (confirming the addition of 4 HAU / 25 μl) and a reference serum titer within twice the expected titer.

[0217] NAI assay Neuraminidase inhibitory (NAI) antibody titers were determined using the method for enzyme-coupled lectin assay (ELLA). Antigen sources (viral NA) were titrated, and standard volumes were selected for incubation with serial dilutions of serum. Serum titration was performed using serial dilutions of serum (heat-inactivated at 56°C for 1 hour), and standard volumes of virus were added in two ways to the wells of a fetuin-coated plate. This mixture was then incubated overnight (16–18 hours); the following day, HRP-conjugated peanut agglutinin PNA (diluted to 2.5 μg / ml) was added to a washed plate and incubated at room temperature for 2 hours. The substrate (ODP in sodium citrate) was added and incubated for 10 minutes to express color. The reaction was then stopped by adding stop buffer (1N sulfuric acid). The plates were scanned for absorbance at OD490 nm. A decrease or absence of color compared to the viral control indicated that NA activity was inhibited due to the presence of NA-specific antibodies. NAI titer (IC)50 The IC50 value was calculated from the OD reading, and the results were graphed in GraphPad Prism. If the ELLA titration curve did not give a good fit to determine a reliable IC50 value, the sample was retested using a different dilution scheme to reach the 50% endpoint.

[0218] T-cell ELISPOT assay Complete medium (DMEM1640 + 10% heat-inactivated FCS) was preheated in a 37°C water bath. PBMCs were rapidly thawed in a 37°C water bath and transferred in droplets to conical tubes with the preheated medium. The tubes were centrifuged at 1,500 rpm for 5 minutes, the cells were resuspended, and counted using a Guava cell counter. Monkey IFN-γ ELISPOT kit (Mabtech 3421M-4APW) and IL-13 ELISPOT kit (Mabtech 3470M-4APW) were used. Pre-coated plates provided by the kits were washed four times with sterile PBS and blocked in 200 μl of complete medium in a 37°C incubator for at least 30 minutes. Sing16 H3 peptide pool (Genscript Custom Order) (each peptide at 1 μg / ml) was used as the recall antigen in the assay. ConA (Sigma CAT#C5275) 2 μg / ml was used as a positive control. 50 μl of recall antigen and 300,000 PBMCs in 50 μl were added to each well for stimulation. The plates were placed in a 37°C, 5% CO2 humidified incubator for 48 hours.

[0219] After incubation, cells were removed, the plate was washed five times with PBS, and 100 μl of 1 μg / ml biotinylated anti-IFN-γ or anti-IL-13 detection antibody was added to each well of the plate. After incubation for 2 hours, the plate was washed five times with PBS and incubated at room temperature for 1 hour with 100 μl of 1:1000 dilution streptavidin in each well. The plate was developed with 100 μl of BCIP / NBT substrate solution until spots appeared. The plate was rinsed with tap water, air-dried, scanned, and counted using a CTL ImmunoSpot® reader (Cellular Technology Ltd.). Data were reported as spot-forming cells (SFCs) per million PBMCs.

[0220] Memory B cell (MBC) ELISPOT assay Using the Human IgG Single-Color Memory B Cell ELISPOT Kit (CAT#NC1911372, CTL) according to the manufacturer's instructions, Sing16 H3-specific and whole IgG + Antibody-secreting cells (ASCs) were measured. Differentiation of MBCs into ASCs was performed in PBMCs using the stimulation cocktail provided by the kit. Briefly, frozen PBMCs were rapidly thawed in a 37°C water bath, mixed with DNase I (CAT#90083, Fisher Scientific), and transferred to tubes containing preheated complete medium (CM) (RPMI 1640, (CAT#22400-089, Gibco)) containing 10% FCS (CAT#SH30073.03, HyClone®) and 1% penicillin / streptomycin (CAT#P4333, Sigma), and centrifuged at 1,500 rpm for 5 minutes. The cell pellet was transferred to 5 ml of complete medium at a rate of 2 × 10⁶ cells per ml. 6Resuspended in cells and transferred to a T25 flask in a 37°C, 5% CO2 incubator for 1 hour. Next, the volume of the cell suspension was adjusted to 6 ml, and B-Poly-S was added at a dilution of 1:1000. The cells were left in the CO2 incubator for 4 days of stimulation. The PVDF microplate supplied by the kit was pre-wetted with 70% ethanol, rinsed, and coated overnight with 80 μl / well of the anti-human IgG capture Ab or Sing16 / H3 recombinant protein 4 μg / ml provided by the kit.

[0221] The cells were harvested 4 days after stimulation, washed, counted, and adjusted to the concentration indicated in the CM. The coated microplate was washed with PBS, blocked with CM for 1 hour, and the contents were transferred. A 100 μl / well cell suspension system was added to the plate and incubated in a CO2 incubator at 37°C for 18 hours. After washing, 80 μl / well of a 1:400 diluted anti-human IgG biotin detection antibody was added to the plate and incubated at room temperature for 2 hours. Following washing, streptavidin-AP diluted at 1:1000 was added to the plate at 80 μl / well for 1 hour. Freshly prepared substrate solution was added and incubated at room temperature for 18 minutes. The plate was rinsed with tap water, air dried, scanned, and counted using a CTL ImmunoSpot® reader (Cellular Technology Ltd.). In each individual animal, the number of IgG + and the number of Sing16 / H3-specific ASCs were calculated per 1 million PBMCs. The frequency of antigen-specific ASCs was calculated as the percentage of antigen-specific ASCs relative to all IgG + ASCs. To evaluate the assay background, negative control wells on all plates were coated with PBS (no background was detected).

[0222] Statistical analysis To estimate the radiant T max non-parametric methods were used to estimate the T of each individual subject based on the observed data. To estimate the half-life of the radiant, to the maximum value max ​Assuming an exponential decay model for luminosity after reaching its target, a linear model was fitted to logarithmically transformed data for each subject over time during decay from maximum luminosity to the baseline (we estimate the baseline using the mean luminosity in the saline group). Half-life was estimated as the point at which logarithmic luminosity reached the midpoint between the maximum and baseline values. In the analysis of different readout information using results summarized as geometric mean, SE model-based geometric mean and SE were estimated from a mixed-effects model for repeated measures when the response was logarithmically transformed readout information, vaccination was a fixed effect, and time was a repeated measure; then, the logarithmically based mean and SE estimates from the model were transformed back to obtain the geometric mean and SE. For weight change, over-descriptive statistical analysis was used. The median and range of maximum % weight loss over time from the baseline (day 0) for each group were reported to assess worse-case scenarios; the median and range of % weight change from the baseline at the last observation for each group were reported to assess weight recovery.

[0223] antigen sequence The sequence of the Perth09 N2 antigen used here is: MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKLAEYRNWSKPQCDITGFAPFSKDNSIRLSAGGDIWVTRE PYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSKEILRTQESECVCINGTCT VVMTDGSASGKADTKILFIEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAFDDGNDVWMGRTISEKSRLGYETFKVIEGWSNPKSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI*(Sequence ID 4) That is the case.

[0224] The sequence of the Mich15 N1 antigen used here is: MNPNQKIITIGSICMTIGMANLQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIRE PFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSC FTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK*(Sequence ID 5) That is the case.

[0225] The sequence of the Sing16 H3 antigen used here is: MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVTQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPALNVTMPNKEQFDKLYIWGVHH PGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKHSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIESIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI*(Sequence ID 6) That is the case.

[0226] The sequence of the Sing16 N2 antigen used here is: MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVTRE PYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLIDSVVSWSKDILRTQESECVCINGTCT VVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAFDDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADLNLMHI* (Sequence ID 7) That is the case.

[0227] The sequence of the CA09 H1 antigen used here is: *(Sequence ID 24) That is the case.

[0228] The sequence of the HA strain A / California / 7 / 2009(H1N1)(CA09) antigen mRNA open reading frame (ORF) used here is: AUGAAAGCUAUCCUGGUCGUCUUGCUGUAUACUUUCGCCACUGCCAACGCCGACACCCUGUGUAUCGGUUACCACGCGAACAACUCCACCGACACUGUGGACACCGUGCUCGAAAAGAAC That is the case.

[0229] The sequence of the A / Michigan / 45 / 2015 (Mich15) neuraminidase (NA) antigen mRNA open reading frame (ORF) used here is: AUGAACCCAAACCAGAAAAUCAUCACGAUUGGCUCGAUUUGCAUGACCAUUGGAAUGGCGAACCUUAUCCUCCAAAUUGGCAACAUUAUCUCGAUCUGGGUCAGCCACUCGAUCCAGAUC GGCAACCAAUCCCAGAUUGAAACUUGCAACCAGAGCGUGAUUACUUACGAAAACAACACGUGGGUGAACCAGACUUACGUCAAUAUUAGCAACACUAACUUCGCCGCUGGGCAGAGCGUC That is the case.

[0230] The sequence of the A / Singapore.INFIMH160019 / 2016(Sing16;H3N2)HA hemagglutinin antigen mRNA open reading frame (ORF) used here is: AUGAAAACCAUAAUCGCGCUCUCAUACAUACUUUGCCUGGUCUUUGCCCAAAAGAUCCCUGGCAACGACAACUCAACCGCGACCCUUUGCCUCGGCCAUCACGCCGUGCCGAACGGCACUAUCGUCAAGACCAUCACAAACGACCGCAUC GAAGUGACCAACGCGACUGAGCUAGUGCAGAACUCCAGCAUUGGAGAGAUUUGCGAUUCUCCACACCAAAUCCUGGACGGAGAGAAUUGUACCUUGAUCGACGCGCUGCUGGGGGAUCCGCAGUGCGACGGAUUCCAGAACAAGAAAUGG GACCUUUUCGUGGAACGGAGCAAGGCAUACUCGAAUUGCUACCCCUACGAUGUGCCCGACUACGCCUCGCUGCGGUCCUUGGUCGCUUCCUCCGGGACCCUGGAAUUCAAAAACGAGAGCUUUAAUUGGACCGGAGUGACCCAGAAUGGC ACCUCGAGCGCCUGCAUUCGGGGCUCCUCCUCGAGCUUCUUCAGCCGCCUGAACUGGCUCACUCACCUCAACUACACCUACCCGGCACUGAACGUGACCAUGCCGAACAAGGAACAAUUCGACAAGCUCUACAUUUGGGGGUGCAUCAC is as follows.

[0231] The sequence of the Perth / 16 / 2009 (H3N2) NA antigen mRNA open reading frame (ORF) used herein is AUGAACCCUAACCAGAAGAUCAUCACAAUUGGAAGCGUGUCCCUGACCAUUUCGACGAUUUGCUUCUUCAUGCAAAUCGCGAUCUUGAUUACCACCGUCACCCUGCAUUUCAAGCAAUACGAAUUCAACUCCCCGCCAAACAACCAAGUCAUGCUCUGCGAGCCCACCAUCAUCGAACGCAACAUCACCGAGAUCGUGUACCUUACCAACACUACCAUCGAAAAGGAGAUUUGCCCCAAGUUGGCCGAAUACCGGAACUGGAGCAAGCCCCAGUGUGACAUCACGGGAUUUGCGCCAUUCAGCAAGGAUAACUCGAUCAGACUUUCCGCCGGGGGCGACAUUUGGGUCACUCGGGAGCCUUACGUGAGCUGCGACCCGGACAAGUGCUACCAAUUCGCACUCGGACAGGGUACCACCCUGAACAACGUCCAUAGCAACAACACCGUGCGCGAUAGAACCCCGUACCGCACCCUCCUCAUGAACGAACUGGGAGUGCCGUUCCACUUGGGAACCAAACAAGUCUGCAUUGCAUGGUCCUCCUCCUCCUGCCACGACGGCAAAGCCUGGCUUCACGUUUGCAUCACCGGCGACGACAAGAAUGCGACGGCCUCCUUCAUAUACAAUGGUAGACUCGUGGAUAGCGUGGUGUCAUGGUCCAAGGAAAUUCUCAGGACUCAGGAGUCAGAGUGCGUGUGCAUCAACGGGACUUGCACUGUCGUG (Sequence ID 11) That is the case.

[0232] The sequence of the A / Wisconsin / 588 / 2019 antigen mRNA open reading frame (ORF) used here is: (Sequence ID 12) That is the case.

[0233] The sequence of the A / Tasmania / 503 / 2020 antigen mRNA open reading frame (ORF) used here is: AGUUUAGCGAGGUGGAGGGCCGCGUGCAGGAUCUGGAAAAGUACGUUGAAGACACCAAGAUCGACCUGUGGUCAUACAAUGCAGAGCUGCUCGUUGCCCUGGAAAAUCAGCACACAA UUGACCUUACAGACUCCGAAAUGAAUAAGCUCUUUGAAAAGACCAAGAAGCAGCUGCGCGAGAACGCCGAGGAUAUGGGGAACGGUUGUUUUAAGAUCUACCACAAGUGUGACAACG CCUGCAUUGGGUCCAUCCGAAAUGAAACAUACGACCACAACGUGUAUAGAGAUGAGGCCCUGAACAACCGAUUCCAGAUUAAGGGAGUCGAGCUGAAGAGUGGCUAUAAGGACUGGA UCCUGUGGAUCUCAUUCGCCAUGUCAUGCUUCCUUCUGUGUAUUGCUCUGCUCGGCUUCAUCAUGUGGGCUUGCCAGAAAGGCAAUAUCCGGUGCAACAUCUGCAUCUAA (SEQ ID NO: 13) That is the case.

[0234] The sequence of the B / Washington / 02 / 2019 antigen mRNA open reading frame (ORF) used here is: AAAUUGAGUUGGCCGUGCUGCUCAGCAACGAAGGCAUAAUCAACAGCGAGGACGAGCACCUCCUGGCUCUGGAGAGAAAGCUGAAGAAGAUGCUCGGCCCUAGCGCAGUUGAGAUCGGAAACGGCUGCUUCGAAACCAAGCACAAGUGCAACCAGACCUGCCUGGACAGGAUCGCGGCAGGAACAUUCGACGCUGGGG AAUUCAGCCUCCCCACCUUCGACAGCCUGAACAUCACAGCCGCCAGUCUGAAUGAUGACGGACUGGAUAACCAUACCAUCCUGCUGUACUACUCUACCGCUGCUUCCUCCCUGGCCGUGACAUUGAUGAUCGCAAUCUUUGUGGUUUAUAUGGUGAGCCGAGACAACGUCAGUUGCAGUAUCUGCCUUUAA (SEQ ID NO: 14) That is the case.

[0235] The sequence of the B / Phuket / 3073 / 2013 antigen mRNA open reading frame (ORF) used here is: AGGAAACUCAAGAAGAUGCUGGGCCCCUCCGCAGUGGACAUUGGGAACGGCUGUUUCGAAACCAAGCAUAAGUGUAACCAGACUUGUCUGGAUAGGAUCGCAGCAGGAACCUUCAACGCCGGCGAAUUUUCUCUGCCAACAUUUGACCUCCCUGAACAUCAC AGCUGCAUCCCUGAACGACGACGGACUGGACAAUCACACCAUCCUGCUGUACUACUCUACUGCCGCUAGCUCCCUGGCCGUGACCCUGAUGCUGGCCAUCUUCAUCGUGUACAUGGUUUCCAGGGAUAACGUGUCUUGUAGCAUUUGCCUGUAA (SEQ ID NO: 15) That is the case.

[0236] result mRNA antigen production, characterization, and expression Full-length codon-optimized HA and NA encoding mRNAs for various influenza strains were enzymatically synthesized using unmodified ribonucleotides. All mRNA preparations had >95% 5' Cap1 and showed a single homogeneous peak on capillary electrophoresis. mRNA-LNP preparations were prepared by mixing various lipid components with mRNA in fixed ratios under controlled conditions. As shown in Table 5, all mRNA-LNPs exhibited >95% encapsulation, with uniform hydrodynamic radii ranging from 95 to 105 nm and polydispersity index (PDI) from 0.060 to 0.136 (Table 5).

[0237] [Table 5]

[0238] Cryo-electron microscopy (cryo-TEM) images of CA09 HA mRNA-LNP showed uniform spherical particles with a multilayered internal core structure. Further analysis of the solid core structure by Fourier transform revealed a 3.7 nm periodicity between layers. The uniform morphology of the particles observed in the micrograph indicates a homogeneous LNP formulation in which the LNPs are properly assembled.

[0239] Antigen expression was confirmed by flow cytometry after transient transfection of human skeletal muscle cells (HskMCs) with unencapsulated mRNA constructs of CA09 HA, Sing16 HA, Sing16 NA, or Mich15 NA, and stained with protein-specific antibodies for analysis. High levels of HA and NA expression were observed from HskMCs, confirming the proper assembly and transport of native HA trimers and NA tetramers upon expression in muscle cells. To study the intracellular localization of expressed HA and NA proteins, HeLa cells were transfected with bivalent H3N2 LNP, and the proteins were visualized by immunostaining and confocal microscopy. When permeabilized cells were stained with antibodies against the corresponding proteins and the endoplasmic reticulum (ER) marker calnexin, the NA signal showed strong co-localization in the ER (approximately 90%), while HA showed moderate co-localization with the ER (25%). This is consistent with the understanding that early NA and HA proteins are moved to the ER for assembly (Dou et al., Front Immunol. (2018) 9:1581).

[0240] The efficiency of mRNA delivery by LNPs and the selection of optimal formulation parameters were evaluated using reporter mRNA expression (Thess et al., Molecular Therapy (2015) 23(1):S55). Single doses of unmodified FF-LNP formulations of 0.05, 0.1, 1, or 5 μg were administered intramuscularly (IM) to mice. Luciferase activity, measured by mean bioluminescence, showed sustained expression from mRNA constructs, peaking at 6 hours post-injection and remaining detectable beyond 72 hours at all doses (Figure 11, panel (a)). High levels of mRNA-mediated protein expression were further validated using hEPO with single doses of 0.1 μg in mice and 10 μg in non-human primates (NHPs). The study was intended to compare LNPs using a standard LNP Dlin-MC3-DMA25 formulation as a control. Serum hEPO, quantified by ELISA, showed peak expression at 6 hours, and erythropoietin expressed using hEPO-LNP was approximately 12 times higher than that expressed using hEPO-MC3 (Figure 11, panel (c)). Both hEPO-LNP and hEPO-MC3 showed similar expression dynamics in NHP and were detectable from 6 hours to 72 hours (Figure 11, panel (d)). These results confirm the usefulness of this LNP formulation for efficient mRNA delivery for expression in both in vitro and in vivo settings.

[0241] Immunogenicity of HA(H1, H3) and NA(N1, N2) mRNA-LNPs in mice Natural history and vaccine studies have shown that antibodies against influenza HA and NA possess antiviral function, and that both antigens are considered important for effective influenza vaccines (Krammer et al., Nat Rev Immunol. (2019) 19(6): pp. 383-97). Unmodified CA09 HA-LNP and Sing16 HA-LNP mRNA vaccines were evaluated in BALB / c mice (n=8) using a two-dose regimen in which mRNA-LNP 2, 0.4, 0.08, or 0.016 μg was administered on a 4-week interval schedule. Total IgG response was evaluated by ELISA using recombinant HA (rHA) antigen from the same strain. HA-specific antibodies were detected in all groups after the first dose, but titers peaked on day 42 after the second dose (Figure 12). To measure functional antibodies, hemagglutination inhibition (HAI) response to homologous CA09 and Sing16 strains was evaluated. HAI titers after the first dose were observed in the 2 μg CA09-LNP and Sing16-LNP treatment groups, with GMT values ​​of 160 and 70, respectively, on day 28. However, a more significant increase in HAI titers was observed after the second dose. On day 42, GMT titers were 80 and 2200 in the 0.016 μg and 0.4 μg groups of the CA09-HA-LNP group, and 14 and 100 in the 0.016 μg and 0.4 μg groups of the Sing 16 HA-LNP group (Figure 13).

[0242] Similarly, in the anti-NA response tests, mice were immunized with 2, 0.4, 0.08, or 0.016 μg of Sing16 NA-LNP or Mich15 NA-LNP. Recombinant NA antigen ELISA was performed to evaluate the total IgG response induced by Mich15 NA-LNP or Sing16 NA-LNP formulations. Animals developed a high antibody-binding response after a single dose, and NA-binding antibodies increased significantly at 42 days after the second dose (Figure 14). Enzyme-coupled lectin assay (ELLA) was used as a surrogate for functional antibody titers for neuraminidase inhibitory (NAI) activity against H6N1 or H6N2 chimeric viruses. Two doses of the vaccine substantially increased the functional antibody response compared to a single dose, but strong NAI titers of GMT800 and GMT60, respectively, were recorded 28 days after the first dose even at low doses of 0.016 μg of Mich15 NA-LNP and Sing16 NA-LNP. On day 42, the Sing16 NA-LNP group showed a dose-dependent response, with GMT titers of 900 and 10200 between 0.4 μg and 0.016 μg, respectively, and in the case of Mich15 NA-LNP, the titer exceeded ULOQ. (Figure 15)

[0243] Defense against virus challenges in mice To test the efficacy of mRNA vaccines in a mouse influenza virus challenge model, we IM-inoculated BALB / c mice with CA09 HA-LNP 0.4 μg twice at weeks 0 and 4, along with a negative control group in LNP dilution buffer. HAI titers in vaccine group serum samples on study days 0, 14, 28, 42, 56, 92, and 107 showed a robust immune response with GMTs of 1660 and 1:830 at days 56 and 92, respectively (Figure 16A). On day 93, all mice were administered a four-fold dose (4×LD) of Belgium 09 virus homologous to CA09, which can produce a 50% lethal outcome. 50The mice were challenged intranasally with HA. All vaccinated mice survived the challenge, with no mortality and only mild pathological symptoms characterized by a transient weight loss of less than 5% (Figure 16B). However, the diluted control group mice suffered significant and rapid weight loss, resulting in a high mortality rate (90%) by day 9. These results demonstrate the high efficacy of HA-based MRT formulations in a lethal mouse influenza challenge model.

[0244] To evaluate the protective efficacy of NA-based MRT vaccines, we performed similar challenge experiments in BALB / c mice. Since the Mich15 NA-LNP vaccine induced robust NAI titers after a single immunization in naive mice (Figure 16A), we evaluated single or double dosing regimens of 0.4 or 0.016 μg of Mich15 NA-LNP administered at 4-week intervals. The control group was vaccinated with the same regimen and received either 0.6 μg of hEPO-LNP or diluted buffer. Robust NAI titers were observed after a single dosing, with GMTs of 14,000 NAI for 0.4 μg of Mich15 NA-LNP and 1,800 NAI for 0.016 μg recorded on day 28 (Figure 17A). After the second immunization on day 42, NAI titers rose to 108,000 NAI in the 4 μg group and 37,000 NAI in the 0.016 μg group. More than 12 weeks after the vaccination regimen, all groups received 4 × LD. 50The mice were challenged with the Belgium 09 H1N1 virus. Individual weight changes over time from the baseline in the treatment groups are graphically shown in Figure 17B. All mice in both control groups suffered significant disease, and all animals had to be euthanized due to >20% weight loss by 8 days after infection. Notably, all animals except one in the vaccine group survived the challenge in the single 0.016 μg dose group, demonstrating high protective efficacy against mortality even after a single dose of only 0.016 μg of Mich15 NA-LNP. Higher doses (0.4 μg) showed overall higher protection, but NA vaccination was not sufficient to prevent weight loss, in contrast to HA immunization, because the vaccinated animals showed a moderate weight loss of 10% of their initial weight, which is consistent with findings reported for other NA vaccines. Weight recovery was observed in the vaccinated group, resulting in a mean final weight change of 2.7% at the low dose and 4.8% at the higher dose compared to the baseline. Overall, the results demonstrate that a single low-dose MRT NA-LNP vaccine can induce functional antibodies sufficient to block influenza NA activity and provide protection against lethal challenges in mice.

[0245] Immunogenicity of HA(H3)mRNA-LNPs in NHP To evaluate the immunogenicity of mRNA-LNPs in NHPs, dose-range studies were conducted in NHPs using 15, 45, 135, and 250 μg of Sing16 HA-LNPs. After the first immunization, all vaccinated NHPs expressed antibodies reactive to recombinant HA protein as indicated in the ELISA (Figure 18). A further boost in titer was observed after the second dose. Surprisingly, the ELISA titer induced by the 15 μg dose was only 1 / 1.8 of the 135 μg dose level (95% CI 1.0, 3.6), suggesting that dose saturation was close to the 15 μg level. Robust HAI antibodies were found in 42 On day 1, the induced and recorded GMTs in all dose groups were 400 for 15 μg, 700 for 45 μg, 900 for 135 μg, and 570 for 250 μg. On day 42, the increase in GMT titer with a 95% CI was 2.2 times (1.0; 5.0) for the 135 μg to 15 μg groups and 1.3 times (0.6; 2.8) for the 135 μg to 45 μg groups, indicating that although a tendency for titer to increase with increasing dose was observed, the difference between groups was minimal (Figure 19A). Neutralizing power, as assessed by microneutralization (MN) assay (Figure 19B), showed a tendency toward a dose-response, with GMTs of 40 for 15 μg, 180 for 45 μg, and 300 for 135 μg on day 28.

[0246] Since T cells have been shown to be effective in reducing viral load and limiting disease severity in animal models (Rimmelzwaan et al., Vaccine (2008) 26(4): D41~D44; Sridhar et al., Nat Med. (2013) 19(10): pp. 1305~12; Sridhar et al., Front Immunol. (2016) 7: p. 195), we evaluated recall T cells in NHP vaccinated with Sing16 HA-LNP 45, 135, 250 μg or recombinant HA 45 μg. PBMCs collected on day 42 were evaluated in IFN-γ (Th1 cytokine) and IL-13 (Th2 cytokine) ELISPOT assays using recall stimulation with pooled overlapping peptides spanning the entire Sing16 HA sequence. All vaccinated animals except one in the 250 μg group expressed IFN-γ secreting cells, ranging from 28 to 1328 spot-forming cells (SFCs) per million PBMCs (Figure 20A). Notably, no dose-response was observed, with both the lower and higher dose levels showing comparable IFN-γ secreting cell frequencies. In contrast, all animals in the control group immunized with recombinant Sing16 HA protein showed the absence of IFN-γ producing cells. The presence of IL-13 cytokine secreting cells was undetectable or very low in all groups tested (Figure 20B). The data suggest that Sing16 HA-LNP induced a potent Th1-biased cell response in NHP, comparable to the cellular response observed with the SARS-CoV-2 vaccine currently under investigation, MRT5500 (Kalnin et al., see above).

[0247] To investigate the frequency of memory B cells (MBCs) in NHP after immunization with Sing16 HA-LNP, the ELISPOT assay was developed to quantify antigen-specific MBCs as information for humoral immunological memory retrieval. On day 180, PBMCs were collected from NHP immunized with Sing16 HA mRNA-LNP 45 μg or 15 μg formulations, or with recombinant HA as a comparator at a 45 μg dose. Four-day polyclonal stimulation of PBMCs, optimized to migrate memory B cells to antibody-secreting cells (ASCs), was performed, and the stimulated PBMCs were seeded on antigen-specific ELISPOT assays, where the frequency of antigen-specific ASCs could be determined. Next, antigen-specific memory B cells were quantified as a percentage of total IgG+ memory B cells. Antigen-specific memory B cells were detected in all animals, with frequencies ranging from 1–5% in the 45 μg dose group and 0.3–1.5% in the 15 μg dose group. In rHA-immunized animals, the memory B cell response appeared to be significantly lower, as antigen-specific memory B cells were undetectable in 5 out of 6 animals (Figure 21). It was concluded that Sing16 HA-LNP, like other mRNA vaccines, induces a population of anti-HA-specific memory B cells that have the potential to extend herd immunity (Lindgren et al., Front Immunol. (2019) 10:614).

[0248] Polyvalent influenza virus antigen The advantage of the mRNA-LNP platform is the flexibility of LNP encapsulation for multiple mRNA antigen constructs. However, this potential needs to be tested to address concerns about antigenic interference. To investigate influenza antigen combinations, H3H1, H3 HA and NA mRNA were co-encapsulated into LNPs, either as bivalent formulations containing 0.2 μg of each mRNA in N2 or N1N2 combination, or as monovalent formulations containing 0.2 μg of each corresponding antigen. These formulations were administered to mice, and the functional titer of each antigen was compared between the bivalent and monovalent formulations to determine the interference of any antigen with immunogenicity (Figure 22, panels (a) to (c) and Table 6).

[0249] [Table 6]

[0250] In the H1H3 combo, no statistically significant difference (p=0.2584) was observed in HAI titers between the co-encapsulated vaccine and the separately administered vaccines at any time point, and no significant difference (p=0.8389) was observed in H3 titers at day 42. In the H3N2 combo, the NA component of the vaccine, in combination with the HA component, induced high neutralizing antibodies, demonstrating no HA dominance. No statistically significant difference (p=0.2960) was observed in H3 titers between the co-encapsulated vaccine and the separately administered vaccines at any time point, and no significant difference (p=0.0904) was observed in N2 titers at day 42. Similarly, in the N1N2 combo, there was no statistically significant difference (p=0.3899) for N2. The N1 titers at day 42 for the co-encapsulated vaccine and the separately administered vaccines were above the limit of quantification. Therefore, combinations of N2N1, H3H1, or H3N2 produced antibody titers equivalent to those of individually formulated LNPs.

[0251] Further investigations were conducted on tetravalent formulations of co-encapsulated H1, N1, H3, and / or N2 mRNA. These formulations, totaling 10 μg, consisting of 2.5 μg of each influenza antigen mRNA and, if necessary, non-coding mRNA (nc mRNA) in combination, were tested in NHP and produced tetravalent (H1N1H3N2), bivalent (H1N1 or H3N2), or monovalent (H1, H3, N1, or N2) LNPs (Table 7).

[0252] [Table 7]

[0253] HAI titers for H1 or H3, or NAI titers for N1 or N2, were compared between monovalent and bivalent or tetravalent formulations (Figure 23). On day 42, the HAI titer for H1 in the tetravalent group was comparable to that of the H1 monovalent group (p=0.9054, t-test, independent test, two-tailed test) or the H1N1 bivalent group (p=0.8002). Similarly, the H3 HAI titer in the tetravalent group was comparable to that of the H3 monovalent group (p=0.2504) or the H3N2 bivalent group (p=0.5894). NAI titers for N1 were nearly identical in groups of animals vaccinated with N1 monovalent mRNA, H1N1 bivalent mRNA, or tetravalent H1N1H3N2 mRNA formulations. Similarly, there was no difference in N2 NAI titer between monovalent N2 mRNA (p=0.8485) or bivalent H3N2 mRNA (0.4545) and tetravalent H1N1H3N2 mRNA preparations.

[0254] Overall, these findings indicate that co-encapsulated or polyvalent vaccines of HA / NA mRNA-LNP at this dose level can deliver all four antigens without concerns about antigen interference, and that all antigens were as immunogenic as they would be in formulations delivered individually. [Examples]

[0255] Additional LNP preparations Additional LNP formulations for mRNA vaccines were prepared and named Lipid C (containing the cationic lipid GL-HEPES-E3-E10-DS-3-E18-1), Lipid D (containing the cationic lipid GL-HEPES-E3-E12-DS-4-E10), and Lipid E ​​(containing the cationic lipid GL-HEPES-E3-E12-DS-3-E14). Human erythropoietin (hEPO) mRNA was used as test mRNA. hEPO expression was measured by ELISA from samples taken from mice injected with the LNPs. Samples were taken at 6, 24, 48, and 72 hours after injection. As shown in Figure 24, hEPO expression was consistently higher at all time points in LNP formulations Lipid A, Lipid B, Lipid C, Lipid D, and Lipid E ​​compared to the control LNP formulation containing the cationic lipid MC3.

[0256] Table 8 below summarizes the results compared to a control LNP containing MC3 cationic lipids. .

[0257] [Table 8]

[0258] Next, the same hEPO mRNA-LNP formulations were tested in non-human primates (NHPs). Samples were collected at 6, 48, and 96 hours after injection. As shown in Figure 25, each LNP formulation produced levels of hEPO comparable to the MC3 control formulation.

[0259] Influenza HA-coding mRNA-LNP preparations were also tested in NHP. NHP administered the LNP preparation at a dose of 10 μg by intramuscular injection, and samples were collected on days 28 and 42 post-injection. HAI titers were measured as described above. As shown in Figure 26, each LNP preparation produced HAI titers comparable to or higher than those of the MC3 control preparation.

[0260] The same experiment as shown in Figure 26 was performed, and HAI titers were measured using Cal09 H1 influenza antigen. As shown in Figure 27, each LNP formulation produced HAI titers comparable to or higher than those of the MC3 control formulation.

[0261] As shown in Figure 28, HAI titers using the Sing16 H3 antigen were elevated in LNP preparations lipid C and lipid D. [Examples]

[0262] Respiratory multinuclear virus (RSV) F protein-coding mRNA LNP preparation The effects of different cationic lipids in LNPs were tested on LNP-encapsulated RSV F protein mRNA. LNP formulations containing lipids A, B, C, D, and E were tested. Each LNP consisted of 40% of one of five cationic lipids, 30% phospholipid DOPE, 1.5% pegylated lipid DMG-PEG2000, and 28.5% cholesterol. LNPs containing the cationic lipid MC3 were also used and considered an industrial benchmark (Jayaraman et al., Angew Chem Int Ed. 51:85 pp. 29-33, 2012).

[0263] The tested F protein was named FD3 and is identical to the pre-fusion RSV F protein. The amino acid sequence of FD3 is listed below.

[0264] FD3 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYID KQLLPILNKQSCSISNPETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKNGSNICLTRTDRGWYCDNAGNVSFFPQAETCKV QSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNELINQSLAFINQSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (Sequence ID 16)

[0265] The mRNA molecules described herein include an open reading frame (ORF) encoding the RSV F protein antigen, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and at least one polyadenylation (Poly(A)) sequence. The mRNA has the following structure: [ka] Further includes a 5' cap with a cap.

[0266] The nucleic acid sequences of the mRNA open reading frame (ORF) encoding the RSV F protein are listed below.

[0267] FD3 mRNA ORF: AUGGAACUGCUGAUCCUCAAAGCCAACGCAAUCACCACCAUUCUCACCGCUGUGACCUUCUGCUUCGCAUCGGGGCAGACAUCACUGAAGAGUUUUACCAGAGCACUUGCACGCGGUGUCAAAGGGUACAUUCCGCACUUGACCUAUGAGCCUCAGCAACAUCAAGGAAAAACUGGACAAUGGCACCGACGCCAAGGUCAAGCUGAUCAACAAGAACUGGACAAGUACAAAGACGCCGGUGACAGAAUUGCAGCCUCUUGAUGGGAUCCGGAACAUCGCGAGGAUGGAUGCAUCCGUGGUCUGUCCAAGGUCUUGCUGCCACCUCGAGGGAAGUAGUCACAAGAUCAAAGAUCAAAGUCCGCGCUGCUGUCAACGAACAAGGCCGUGUCUCUCUCUACGGCGUGCUCUCUCUACGUCUCUCU ACCUGAAAAAUAUACAUGACAAGCAGCUGCUGCCCAUCCUCAACAAGCAAUCCUGCUCCAUCUCCCAACCCCGAAACCGUGAUCGAGUUCCAGCAGAGAAAAACCGCCUGCUGGAAAAUACUCCGGAGUUCUCUGGAAUGCCGGCGUGACCACCCCUGUGUCCACCUAACUGACUGACCAACUCCGAGCUUCUCUCCCUUAUCAAUGACAUGCCUAUCACCGAACGACCAG AAAAGCUGAGUCGAAACGUGCAGAUUGUGGCGGCAGCAGUCAUACAGCAUCAUGCGAUCAUCAAGGAAGAAGUGCUGGCGUACGUGGUGCAACUCCCGCUGUACGGCGUCAUCGAUACCCCGUGCUGGAAGCUGCACACCUCGCCUUUGUACCACCAAACACCAAGAACGGAUCCAACAUCUGCUUAACCCGGGACUGAUCGGGGGUUGGUACUGCGACACGCCGG (Sequence ID 17)

[0268] The nucleic acid sequences of the DNA template encoding the RSV F protein are listed below.

[0269] FD3 DNA: TGTCATGCTACGGAAAGACTAAGTGCACCGCCTCGAACAAGAACCGCGGCATCATTAAGACTTTCTCGAATGGTTGCGACTATGTGTCCAACAAGGGCGTGGATACTGTGTCAGTCGGGAATACTCTTTACTACGTGAACAAGCAGGAGGGGAAAAGCCTCTACGTGAAGGGAGAGCCTATTATCAACTTTTACGATCCGCTGGTGTTCCCGTCCGACGAATTCGACGCCAGCATCAGCCAAGTCA ACGAGCTGATTAACCAGTCCCTCGCCTTCATCAACCAATCCGACGAGCTCCTGCATAACGTGAACGCGGAAAGTCCACCACCAACATCATGATCACTACTATTATCATCGTGATCATCGTCA TCCTGCTGAGCCTGATTGCTGTGGGCCTGTTGCTGTATTGCAAAGCCAGGTCCACCCCGGTCACCCTGTCGAAGGATCAGCTGTCCGGAATCAACAACATTGCCTTCTCCAACTAA (SEQ ID NO: 18)

[0270] The nucleic acid sequences of the 5'UTR and 3'UTR are listed below.

[0271] 5'UTR: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 19)

[0272] 3'UTR: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 20)

[0273] The nucleic acid sequence of the full-length mRNA encoding the RSV F protein is listed below.

[0274] FD3 mRNA: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGAACUGCUGAUCCUCAAAGCCAACGCAAUCACCACCAUUCUCACCGCUGUGACCUUCUGCUUCGCAUCGGGGCAGAACAUCACUGAAGAGUUUUACCAGAGCACUUGCAGCGCGGUGUCAAAGGGUUACCUUUCCGCACUGCGGACCGGAUGGUACACUUCCGUGAUCACCAUUGAGCUCAGCAACAUCAAGGAAAACAAGUGCAAUGGCACCGACGCCAAGGUCAAGCUGAUCAAACAAGAACUGGACAAGUACAAGAACGCCGUGACAGAAUUGCAGCUCCUGAUGGGAUCCGGAAACGUCGGUCUGGGCGGAGCCAUCGCGAGUGGAGUGGCUGUGUCCAAGGUCUUGCACCUCGAGGGAGAAGUGAACAAGAUCAAGUCCGCGCUGCUGUCAACGAACAAGGCCGUGGUGUCCCUGUCUAACGGCGUCAGCGUGCUGACGUUCAAGGUCCUGGACCUGAAGAAUUACAUUGACAAGCAGCUGCUGCCCAUCCUCAACAAGCAAUCCUGCUCCAUCUCCAACCCCGAAACCGUGAUCGAGUUCCAGCAGAAGAACAACCGCCUGCUGGAAAUUACUCGCGAGUUCUCUGUGAAUGCCGGCGUGACCACCCCUGUGUCCACCUACAUGCUGACCAACUCCGAGCUUCUCUCCCUU

[0275] The LNP-RSV FD3 mRNA composition was administered to NHPs. A group of six cynophobic macaques received either a 5 μg dose of the above LNP-encapsulated mRNA or a 10 μg dose of the Al(OH)3-adjuvanted RSV Pre-F NP subunit control vaccine via intramuscular (IM) injection on days 0 and 21. The monkeys were induced to bleed prior to each vaccine administration and two weeks after the last vaccination (D35). As shown in Figure 29, all cationic lipids tested effectively induced the production of anti-RSV F protein antibodies to levels similar to those of Pre-F NP with aluminum adjuvant.

[0276] As shown in Figure 30, all of the cationic lipids tested produced effective RSV neutralization values ​​comparable to those of Pre-F NPs using aluminum adjuvants.

[0277] The cumulative results for Figures 29 and 30 are shown in Tables 9 and 10 below.

[0278] [Table 9]

[0279] [Table 10]

[0280] A better quality immune response is indicated by a lower antibody titer / neutralization titer ratio. Here, LNP formulation lipid B showed the best quality immune response, and all LNP formulations showed a superior quality immune response compared to non-mRNA vaccines and Pre-F NPs, with some even outperforming the MC3 industrial benchmark LNP formulation. [Examples]

[0281] SARS-CoV-2 spike (S) protein-coding mRNA LNP preparation LNP preparations containing SARS-CoV-2 spike (S) protein-coding mRNA Human subjects were administered an LNP preparation containing SARS-CoV-2 S protein-coding mRNA. Subjects received LNP of preparation lipid B. The unmodified mRNA encoded a SARS-CoV-2 S protein that had been mutated by removing the furin cleavage site and replacing residues 986 and 987 with proline. Subjects received the LNP-SARS-CoV-2 vaccine under the clinical trial protocol for NCT04798027 described below.

[0282] This was a sequential-group prevention study consisting of a sentinel cohort followed by a fully registered cohort. The sentinel cohort had three dose levels (up to 25 participants aged 18–49 years for each dose level), and this was conducted in an open-label manner along with a graded safety assessment for each dose level and each vaccine. All sentinel participants received two doses of vaccine 21 days apart. The fully registered cohort was stratified into two age groups based on age at registration: a young adult age group (140 participants aged 18–49 years) and an older age group (168 participants aged ≥50 years). Fully registered cohort 1 (groups 1–4) received a single injection study intervention, while participants in cohort 2 (groups 5–8) received two doses of vaccine (administered 21 days apart). The route of administration was determined for all groups. It was intramuscular (IM).

[0283] Study: Group 1 - One injection of SARS-CoV-2 mRNA vaccine formulation 1 on day 1.

[0284] Study: Group 2 - One injection of SARS-CoV-2 mRNA vaccine formulation 2 on day 1.

[0285] Study: Group 3 - One injection of SARS-CoV-2 mRNA vaccine formulation 3 on day 1.

[0286] Placebo comparator: Group 4 - One injection of placebo (0.9% saline) on day 1.

[0287] Study: Group 5 - Two injections of SARS-CoV-2 mRNA vaccine formulation 1 on days 1 and 22.

[0288] Study: Group 6 - Two injections of SARS-CoV-2 mRNA vaccine formulation 2 on days 1 and 22.

[0289] Study: Group 7 - Two injections of SARS-CoV-2 mRNA vaccine formulation 3 on days 1 and 22.

[0290] Placebo comparator: Group 8 - Two injections of placebo (0.9% saline) on days 1 and 22.

[0291] The study results showed that 91%–100% of study participants, across all three tested doses, achieved seroconversion of neutralizing antibodies (defined as a four-fold increase relative to the baseline) two weeks after the second injection. No safety concerns were observed, and the tolerability profile was comparable to other unmodified mRNA SARS-CoV-2 vaccines. [Examples]

[0292] Further research on quadrivalent or octavalent influenza vaccine LNP formulations HAI and NAI titers were measured in mice administered with various polyvalent LNP-influenza mRNA vaccines. HAI titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / Maryland / 15 / 2016 BX69A, and B / Phuket / 3073 / 2013. NAI titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / Colorado / 06 / 201, and B / Phuket / 3073 / 2013.

[0293] HAI and NAI titers were compared in mice receiving monovalent or tetravalent HA or NAI mRNA vaccines.

[0294] Mice were injected with a prime vaccine on day 0 and a booster vaccine of the same dose on day 21. Blood was collected on days 1, 20, 22, and 35. In monovalent compositions containing mRNA encoding HA or NA antigens, mRNA encoding each of the following was used individually: H1, H3, and HA from the B / Victoria lineage, and HA from the B / Yamagata lineage (specifically, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013); and N1, N2, and NA from the B / Victoria lineage, respectively, and from the B / Yamagata lineage. A tetravalent vaccine composition was also prepared containing mRNA encoding NA, as well as H1, H3, and HA from the B / Victoria lineage, respectively, and HA from the B / Yamagata lineage (specifically, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013). Finally, an octavalent vaccine composition was prepared containing mRNA encoding H1, H3, and HA from the B / Victoria lineage, HA from the B / Yamagata lineage, N1, N2, and NA from the B / Victoria lineage, and NA from the B / Yamagata lineage (specifically, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013), and administered as an octavalent vaccine. For all compositions, each mRNA was added at a dose of 0.4 μg / strain. Each group consisted of n=6 mice.

[0295] The outlines of each experimental group are listed in Table 11 below.

[0296] [Table 11]

[0297] As shown in Figure 31, the octavalent mRNA-LNP preparation produced HAI titers within four times that of the tetravalent preparation for three of the four influenza strains.

[0298] A summary of the NAI titer results for each of the above groups is shown in Figure 33. The octavalent mRNA-LNP preparation produced NAI titers comparable to those of the tetravalent mRNA-LNP preparation.

[0299] Thus, the data demonstrate that the octavalent vaccine was able to induce robust HA and NA immune responses, and that the presence of immunodominant HA derived from four different influenza strains does not appear to suppress or interfere with the anti-NA immune response.

[0300] High-content imaging-based neutralization assay (HINT) titers for HA and NAI titers were further measured in ferrets administered with various polyvalent LNP-influenza mRNA vaccines. The HINT assay is described in more detail in Jorquera et al. (Scientific Reports. 9: 2676, 2019), which is incorporated herein by reference. HINT titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / IOWA / 06 / 2017, and B / Phuket / 3073 / 2013. NAI titers were measured against influenza strains A / Michigan / 45 / 2015, A / SINGAPORE / INFIMH160019 / 2016, B / Colorado / 06 / 2017, and B / Phuket / 3073 / 2013.

[0301] Ferrets used to evaluate the immunogenicity of the multivalent vaccine were vaccinated twice, 21 days apart, with either (1) a mixture of four mRNAs encoding the NA antigen (N1, N2, BvNA, and ByNA), (2) a mixture of four mRNAs encoding the HA antigen (H1, H3, BvHA, and ByHA), or (3) a mixture of four mRNAs encoding the NA antigen (N1, N2, BvNA, and ByNA) and four mRNAs encoding the HA antigen (H1, H3, BvHA, and ByHA), as shown in Table 12 below. Each HA contains HA derived from one of the following four strains: A / Michigan / 45 / 2015(H1); A / Singapore / Infimh-16-0019 / 2016(H3); B / Iowa / 06 / 2017(B / Victoria lineage); and B / Phuket / 3073 / 2013(B / Yamagata lineage). All antigens were administered in a 1:1 ratio.

[0302] The outlines of each experimental group are listed in Table 12 below.

[0303] All ferrets were induced to bleed under sedation (isoflurane) as required, at baseline, one day before or immediately before booster, at booster vaccination, and two weeks after challenge. Serum samples (stored at -20°C until needed) were tested by ELLA to assess NAI activity. Furthermore, hemagglutination inhibition assays (HAIs) were initiated to evaluate the antibody response to hemagglutinin antigens following multivalent vaccination.

[0304] [Table 12]

[0305] A summary of the HINT results for each of the above groups is shown in Figure 32. The octavalent mRNA-LNP preparation produced HINT titers comparable to those of the tetravalent mRNA-LNP preparation.

[0306] A summary of the NAI titer results for each of the above groups is shown in Figures 34 (Day 20) and 35 (Day 42). The octavalent mRNA-LNP preparation produced NAI titers comparable to those of the tetravalent mRNA-LNP preparation. This was true for samples from Day 20 to Day 42. [Examples]

[0307] Functional antibody titers against heterologous influenza subtype strains recorded using mRNA in lipid A or lipid B LNP preparations. To evaluate the immunogenicity of mRNA-LNPs in NHP, 0, 15, and 45 μg of Sing16 HA-coding mRNA (encoding HA A / Singapore / INFIMH-16-0019 / 2016) encapsulated in lipid A or lipid B LNP preparations were administered. Naive male and female crab-eating macaques (Macaca fascicularis) originating from Mauritius were used. At the start of the study, the animals weighed >2 kg and were >2 years old. Each group consisted of a maximum of 6 animals, and on study day 0, 0.5 mL of their respective vaccine dose or dilution was administered via the IM route to one forelimb of each animal, targeting the deltoid muscle. A second immunization was administered to the opposite limb of the animal 28 days after the first immunization occurred. A tetravalent oocyte-derived inactivated influenza vaccine (IIV) containing the A / Singapore / INFIMH-16-0019 / 2016(H3N2) strain was used as a comparator.

[0308] Influenza assays were performed using the A / Singapore / INFIMH-16-0019 / 2016(H3N2) virus stock from BIOQUAL, Inc. Additional width testing by HAI was performed using the following H3N1 virus stocks: A / Shandoglaicheng / 1763 / 2016, A / Louisiana / 13 / 2017, A / Kenya / 105 / 2017, A / Victoria / 746 / 2017, and A / Michigan / 84 / 2016, A / Aksaray / 4048 / 2016. These include strains derived from both the 3c.2a and 3c.3a clades, as well as a very distantly related porcine-like H3 sequence (A / Michigan / 84 / 2016) based on bioinformatics analysis to select the most diverse set of H3N2 sequences from the same timeframe as A / Singapore / INFIMH-16-0019 / 2016.

[0309] In the microneutralization (MN) assay, serum samples were diluted with receptor-disrupting enzyme (Denka Seiken, 370013) and incubated overnight in a 37°C water bath. The samples were then thermally inactivated at 56°C for 30 minutes, followed by two sets of 2-fold serial dilutions in 96-well plates. 100TCID 50 Equivolute amounts of virus were added to plates and incubated at 37°C for 1 hour. 100 microliters of the sample / virus mixture was transferred to a 96-well flat-bottom plate of MDCK cells (ATCC#CCL-34) containing TPCK-treated medium and incubated at 37°C for 48 hours with 5% CO2. The plates were fixed with cold acetone, then stained with biotin-conjugated anti-influenza A NP (Millipore, MAB8258B), and subsequently incubated with DELFIA europium-labeled streptavidin in Delfia assay buffer. Fluorescence was measured and endpoint titers were reported.

[0310] On day 43, after the second immunization, NHPs vaccinated with Sing16HA-CL-059 and Sing16HA-CL017 expressed neutralizing antibodies against the homologous virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2), as described in the MN assay (Figures 36 and 37). Furthermore, in this model, contrary to the IIV vaccine, A / Shandoglaicheng / 1763 / 2016, A MN titers were observed in a heterogeneous subtype virus panel including / Louisiana / 13 / 2017, A / Kenya / 105 / 2017, A / Victoria / 746 / 2017, and A / Aksaray / 4048 / 2016. The data indicate that the mRNA formulation has the potential to offer a wider range than IIV, which substitutes for heterogeneous subtype strains of influenza.

Claims

1. A pharmaceutical composition comprising nucleic acid molecules encapsulated in lipid nanoparticles (LNPs), wherein the LNPs are Cationic lipids in a molar ratio of 35% to 45%, Polyethylene glycol (PEG) conjugate lipids in a molar ratio of 0.25% to 2.75% Cholesterol-based lipids in a molar ratio of 20% to 35%, and It contains helper lipids in a molar ratio of 25% to 35%. Pharmaceutical composition in which all molar ratios are relative to the total lipid content of LNP.

2. LNP is Cationic lipids in a molar ratio of 40%, Pegged lipids at a molar ratio of 1.5%, Cholesterol-based lipids at a molar ratio of 28.5%, and Contains helper lipids at a molar ratio of 30% The composition according to claim 1.

3. The composition according to claim 1 or 2, wherein the cationic lipid is 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.

4. The composition according to any one of claims 1 to 3, wherein the pegylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

5. The composition according to any one of claims 1 to 4, wherein the cholesterol lipid is cholesterol.

6. The composition according to any one of claims 1 to 5, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

7. LNP is, OF-02 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30% The composition according to claim 1, including

8. LNP is, cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30% The composition according to claim 1, including

9. LNP is, GL-HEPES-E3-E10-DS-3-E18-1 in a 40% molar ratio, 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% The composition according to claim 1, including

10. LNP is, GL-HEPES-E3-E12-DS-4-E10 in a 40% molar ratio, 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% The composition according to claim 1, including

11. LNP is, GL-HEPES-E3-E12-DS-3-E14 in a 40% molar ratio, 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% The composition according to claim 1, including

12. The composition according to any one of claims 1 to 11, wherein the LNP has an average diameter of 30 to 200 nm.

13. The composition according to claim 12, wherein the LNP has an average diameter of 80 to 150 nm.

14. The composition according to any one of claims 1 to 13, comprising 1 to 10, optionally 1 mg / mL of LNP.

15. The composition according to any one of claims 1 to 14, wherein the LNP comprises 1 to 20, optionally 5 to 10 or 6 to 8 nucleotide molecules.

16. The composition according to any one of claims 1 to 15, wherein the nucleic acid molecule(s) is an mRNA molecule containing an open reading frame (ORF).

17. The composition according to claim 16, wherein the mRNA molecule encodes an antigen, optionally a viral antigen or a bacterial antigen.

18. The composition according to claim 17, wherein the antigen is derived from the influenza virus.

19. The composition according to claim 17 or 18, wherein the LNP comprises two or more mRNA molecules, each mRNA molecule encoding a different antigen, and the different antigens may be from the same pathogen or from different pathogens.

20. The composition according to claim 17 or 18, comprising two or more LNPs, each LNP comprising mRNA encoding a different antigen, wherein the different antigens may originate from the same pathogen or from different pathogens.

21. The composition according to claim 19 or 20, comprising (i) one or more hemagglutinin (HA) antigens, (ii) one or more neuraminidase (NA) antigens, or (iii) two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding at least one HA antigen and at least one NA antigen.

22. One or more antigens encoding influenza A, B, and / or C viruses The composition according to any one of claims 18 to 21, comprising the above mRNA molecule, wherein the antigen is optionally the HA and / or NA antigen of influenza A and influenza B viruses.

23. The composition according to claim 22, wherein 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; and / or the NA antigen of influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11.

24. The composition according to claim 22 or 23, wherein the HA and NA antigens of the influenza B virus are derived from the influenza B / Yamagata lineage or the influenza B / Victoria lineage.

25. The composition according to any one of claims 22 to 24, comprising (i) one or more HA antigens, (ii) one or more NA antigens, or (iii) two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding one or more combinations of HA antigens and NA antigens selected from the H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H10N7 subtypes and / or the B / Yamagata lineage and the influenza B / Victoria lineage.

26. The composition according to any one of claims 22 to 25, comprising one mRNA molecule encoding an H3 HA antigen, one mRNA molecule encoding an H1 HA antigen, one mRNA molecule encoding an HA antigen derived from the influenza B / Yamagata lineage, and one mRNA molecule encoding an HA antigen derived from influenza B / Victoria.

27. The composition according to any one of claims 22 to 26, comprising one mRNA molecule encoding an H3 HA antigen, one mRNA molecule encoding an N2 NA antigen, one mRNA molecule encoding an H1 HA antigen, one mRNA molecule encoding an N1 NA antigen, one mRNA molecule encoding an HA antigen derived from the influenza B / Yamagata lineage, one mRNA molecule encoding an NA antigen derived from the influenza B / Yamagata lineage, one mRNA molecule encoding an HA antigen derived from the influenza B / Victoria lineage, and one mRNA molecule encoding an NA antigen derived from the influenza B / Victoria lineage.

28. The composition according to claim 16, wherein the mRNA molecule comprises an open reading frame (ORF) encoding the respiratory multinuclear virus (RSV) F protein antigen.

29. The composition according to claim 28, wherein the RSV F protein antigen comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 16 or consists of the amino acid sequence of SEQ ID NO:

16.

30. The composition according to claim 28 or 29, wherein the RSV F protein antigen is a pre-fusion protein.

31. The composition according to any one of claims 16 to 30, wherein the ORF is codon-optimized.

32. The composition according to any one of claims 16 to 31, wherein the mRNA molecule 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.

33. The composition according to any one of claims 16 to 32, wherein the mRNA comprises at least one chemical modification.

34. The composition according to any one of claims 16 to 33, 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 the mRNA are chemically modified.

35. The composition according to any one of claims 16 to 33, 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 the ORF are chemically modified.

36. The composition according to any one of claims 33 to 35, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpsoiduridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazalpsoiduridine, 2-thio-1-methylpsoiduridine, 2-thio-5-azauridine, 2-thio-dihydropsoiduridine, 2-thio-dihydrouridine, 2-thiopsoiduridine, 4-methoxy-2-thiopsoiduridine, 4-methoxypsoiduridine, 4-thio-1-methylpsoiduridine, 4-thiopsoiduridine, 5-azauridine, dihydropsoiduridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

37. The composition according to claim 36, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpsoiduridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

38. The composition according to claim 36, wherein the chemical modification is N1-methylpsoidouridine.

39. The composition according to any one of claims 28 to 38, wherein the mRNA comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence shown in Sequence ID No.

17.

40. The composition according to any one of claims 28 to 38, wherein the mRNA comprises a nucleic acid sequence having at least 80% identity with the nucleic acid sequence shown in Sequence ID No.

21.

41. mRNA has the following structural elements: (i) A 5' cap having the following structure: 【Chemistry 1】 (ii) The 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 19; (iii) Protein coding region having the nucleic acid sequence of Sequence ID No. 17; (iv) the 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 20; and (v) Poly(A) Tail A composition according to any one of claims 28 to 38, comprising:

42. A composition according to any one of claims 1 to 41, formulated for intramuscular injection.

43. The composition according to claim 42, comprising phosphate-buffered saline.

44. The composition according to any one of claims 1 to 43, optionally comprising trehalose in an amount of 10% (w / v) of the composition.

45. To provide an aqueous buffer solution containing nucleic acid molecules, To provide an amphiphilic solution containing cationic lipids, pegylated lipids, cholesterol-based lipids, and helper lipids, and A method for producing the composition according to any one of claims 1 to 44, comprising mixing an aqueous buffer solution and an amphiphilic solution in a ratio of 5:1 to 3:1, and optionally 4:

1.

46. The method according to claim 45, wherein the aqueous buffer solution is, in some cases, an acidic buffer solution containing 1 mM citric acid and 150 mM sodium chloride at a pH of approximately 4.

5.

47. The method according to claim 45 or 46, wherein the amphiphilic solution is an ethanol solution.

48. A method for inducing an immune response in a subject requiring such induction, comprising administering a prophylactic effective amount of the composition according to any one of claims 1 to 47 to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

49. A method for preventing influenza infection or reducing one or more symptoms of influenza infection, comprising administering, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a preventively effective amount of the composition described in any one of claims 18 to 27.

50. The method according to claim 49, wherein the composition induces an immune response to one or more seasonal and / or pandemic influenza strains.

51. The method according to any one of claims 48 to 50, comprising administering to a subject one or more doses of a composition, each dose containing 1 to 250, and optionally 2.5, 5, 15, 45, or 135 μg of mRNA.

52. The method according to any one of claims 48 to 51, comprising administering two doses of the composition to a subject at intervals of 2 to 6, optionally 4 weeks.

53. In some cases, use of the composition according to any one of claims 1 to 44 for the manufacture of a drug for use in treating an object requiring treatment by the method according to any one of claims 48 to 52.

54. A composition according to any one of claims 1 to 44, for use in any case to treat an object requiring treatment by the method according to any one of claims 48 to 52.

55. Depending on the circumstances, the container may be a vial or a pre-filled syringe or injector, comprising a single-use or multi-use dose of the composition according to any one of claims 1 to 44. A kit including a container.

56. The composition according to any one of claims 21 to 27, wherein the antigen comprises an influenza virus HA antigen and / or influenza virus NA antigen having a molecular sequence identified or designed from a machine learning model.

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