Lipid nanoparticles for delivering mRNA vaccines

Abstract

Novel lipid nanoparticles for delivering nucleic acids, such as mRNA, are provided. Methods for making and using lipid nanoparticles for delivering nucleic acids, such as mRNA, are also provided.

Description

[Technical Field]

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

[0002] Messenger RNA (mRNA)-based vaccines offer a promising alternative to traditional subunit vaccines containing pathogen-derived antigenic proteins. These proteins are typically produced recombinantly, requiring bacterial fermentation and / or cell culture and complex purification. mRNA-based vaccines allow for the de novo expression of complex antigens in vaccinated subjects, which in turn allows for appropriate post-translational modifications and presentation of the antigen in its native form. Unlike traditional technologies, mRNA vaccine production does not require complex and expensive bacterial fermentation, tissue culture, and purification methods. Furthermore, once established, mRNA vaccine manufacturing methods can be used for a variety of antigens, enabling the rapid development and distribution of mRNA vaccines. Furthermore, mRNA vaccines are inherently safe delivery vectors because they only transiently express antigens and do not integrate into the host genome. Because the antigens encoded by the mRNA are produced in vivo in the vaccinated individual, mRNA vaccines are particularly effective in eliciting both humoral and T cell-mediated immunity. Summary of the Invention [Problem to be solved by the invention]

[0003] However, RNA is unstable and prone to rapid degradation. It lacks a natural cell surface receptor that facilitates cellular uptake of RNA. Indeed, the development of mRNA has been hampered by the inefficient in vivo delivery of mRNA. Therefore, there remains a need to develop vaccine formulations that can improve mRNA delivery in vivo. [Means for solving the problem]

[0004] The present disclosure provides pharmaceutical compositions comprising a nucleic acid molecule (e.g., an mRNA molecule) encapsulated in lipid nanoparticles (LNPs), each LNP comprising a cationic lipid at a molar ratio of 35% to 45%, a polyethylene glycol (PEG)-conjugated (PEGylated) lipid at a molar ratio of 0.25% to 2.75%, a cholesterol-based lipid at a molar ratio of 20% to 35%, and a helper lipid at a molar ratio of 25% to 35%, all based on the total lipid content of the LNP. The compositions may be used as vaccines to induce immune protection in subjects (e.g., human subjects) in need of such protection.

[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 LNPs comprise a cationic lipid at a molar ratio of 40%, a PEGylated lipid at a molar ratio of 1.5%, a cholesterol-based lipid at a molar ratio of 28.5%, and a helper lipid at 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 LNPs comprise 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 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%.

[0008] In some embodiments, the LNP comprises 1 to 20, 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 an influenza virus antigen, or a bacterial antigen).

[0009] In some embodiments, the LNP comprises two or more mRNA molecules, each encoding a different antigen, optionally the different antigens being from the same pathogen or from different pathogens. In some embodiments, the composition comprises two or more LNPs, each comprising mRNA encoding a different antigen, optionally the different antigens being from the same pathogen or from different pathogens.

[0010] For example, the composition may contain two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding (i) different hemagglutinin (HA) antigens, (ii) different neuraminidase (NA) antigens, or (iii) 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, an 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 to 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 to 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: [ka] (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 19; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO: 17; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 20; and (v) Poly(A) tail.

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

[0026] In another aspect, the present disclosure provides a method for producing an LNP composition herein, comprising providing an aqueous buffer solution containing nucleic acid molecules; providing an amphiphilic solution containing cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids; and mixing the aqueous buffer solution and 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 a pH of about 4.5). The amphiphilic solution may be, for example, an ethanol solution.

[0027] In another aspect, the present disclosure provides methods for eliciting an immune response in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of the LNP composition, 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 given at intervals of 2 to 24, optionally 4, 8, 12, 16, or 20 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0028] Also provided herein is the use of the composition for the manufacture of a medicament for use in treating a subject in need thereof, as well as a composition for use in treating a subject in need thereof.

[0029] The present disclosure also provides kits comprising a container containing a single-use or multi-use dosage of the composition, optionally wherein the container is a vial or a pre-filled syringe or injector.

[0030] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising: Cationic lipids at a molar ratio of 35% to 45% Polyethylene glycol (PEG) conjugated (PEGylated) lipids at molar ratios of 0.25% to 2.75% Cholesterol-based lipids at a molar ratio of 20% to 35%, and Contains helper lipids at a molar ratio of 25% to 35%; All molar ratios are relative to the total lipid content of the LNPs; A pharmaceutical composition is provided that includes an mRNA molecule encapsulated in an LNP, wherein the mRNA molecule includes an open reading frame (ORF) encoding an antigen derived from an influenza virus.

[0031] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising: Cationic lipids at a molar ratio of 35% to 45% Polyethylene glycol (PEG) conjugated (PEGylated) lipids at molar ratios of 0.25% to 2.75% Cholesterol-based lipids at a molar ratio of 20% to 35%, and Contains helper lipids at a molar ratio of 25% to 35%; All molar ratios are relative to the total lipid content of the LNPs; A pharmaceutical composition is provided that includes an mRNA molecule encapsulated in an LNP, wherein the mRNA molecule includes an open reading frame (ORF) encoding a respiratory syncytial virus (RSV) F protein antigen.

[0032] Other features, objects, and advantages of the present invention will become apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of example only, and not of limitation. 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 drawings]

[0033] [Figure 1A] Figure 1A is a pair of graphs showing human erythropoietin (hEPO) expression in mice treated with various LNP formulations of hEPO mRNA. Panel a): LNP formulations "Lipid A" and "Lipid B" compared to MC3. Bars represent the mean and standard deviation. Panel b): Formulations made with cationic lipid OF-02. PEG: DMG-PEG2000. Cholest: Cholesterol. "Lipid A": Unless otherwise indicated, LNP composition containing OF-02, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. "Lipid B": LNP composition containing cKK-E10, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 1B] FIG. 1B is a pair of graphs showing hEPO expression in mice and non-human primates (NHPs) using LNP formulations Lipid A and Lipid B. [Figure 2A]Figures 2A and 2B are a pair of graphs showing that lipid A and lipid B LNP formulations carrying mRNA encoding the hemagglutinin (HA) of strain A / California / 7 / 2009 (H1N1) (CA09) induced robust functional antibodies (Figure 2A) and protected mice from death or emaciation (greater than 20%) when challenged with a pandemic strain of influenza virus (Figure 2B). Hemagglutinin inhibition (HAI) titers are reported as log10 for serum samples collected on study days 0, 14, 28, 42, 56, 92, and 107. Bars represent geometric means and geometric standard deviations. Daily body weights were measured after intranasal challenge (day 93) with 4 LD50 of A / Belgium / 2009 (H1N1) (Belgium09). Body weights are presented as percentages of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting weight and in all mice 14 days post-infection (day 107). rHA: recombinant hemagglutinin. AF03: oil-in-water emulsion adjuvant. Diluent = PBS. LLOQ = lower 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% weight loss clipped relative to the weight on the day of challenge. [Figure 2B]Figures 2A and 2B are a pair of graphs showing that lipid A and lipid B LNP formulations carrying mRNA encoding the hemagglutinin (HA) of strain A / California / 7 / 2009 (H1N1) (CA09) induced robust functional antibodies (Figure 2A) and protected mice from death or emaciation (greater than 20%) when challenged with a pandemic strain of influenza virus (Figure 2B). Hemagglutinin inhibition (HAI) titers are reported as log10 for serum samples collected on study days 0, 14, 28, 42, 56, 92, and 107. Bars represent geometric means and geometric standard deviations. Daily body weights were measured after intranasal challenge (day 93) with 4 LD50 of A / Belgium / 2009 (H1N1) (Belgium09). Body weights are presented as percentages of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting weight and in all mice 14 days post-infection (day 107). rHA: recombinant hemagglutinin. AF03: oil-in-water emulsion adjuvant. Diluent = PBS. LLOQ = lower 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% weight loss clipped relative to the weight on the day of 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 LNPs induced robust functional antibodies (Figure 3A) and protected mice from weight loss and death when challenged with a pandemic strain of influenza virus (Figure 3B). Neuraminidase inhibition (NAI) titers are reported as log10 for serum samples collected on study days 14, 28, 42, 56, 88, and 114. Daily body weights were measured after intranasal challenge with 4 LD50 of Belgium09 (day 89 for the single-dose group or day 117 for the two-dose group). Body weights are presented as percentage of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting weight, and in all mice 14 days post-infection (day 103 for the single-dose group or day 131 for the double-dose group). Bars represent 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 relative to the weight on the day of challenge. Administered mRNA: 0.4 or 0.016 μg of mRNA encoding Mich15 NA. Control: 0.6 μg of mRNA encoding hEPO or diluent (PBS). [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 LNPs induced robust functional antibodies (Figure 3A) and protected mice from weight loss and death when challenged with a pandemic strain of influenza virus (Figure 3B). Neuraminidase inhibition (NAI) titers are reported as log10 for serum samples collected on study days 14, 28, 42, 56, 88, and 114. Daily body weights were measured after intranasal challenge with 4 LD50 of Belgium09 (day 89 for the single-dose group or day 117 for the two-dose group). Body weights are presented as percentage of weight loss from the day of challenge. Euthanasia occurred in mice that lost more than 20% of their starting weight, and in all mice 14 days post-infection (day 103 for the single-dose group or day 131 for the double-dose group). Bars represent 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 relative to the weight on the day of challenge. Administered mRNA: 0.4 or 0.016 μg of mRNA encoding Mich15 NA. Control: 0.6 μg of mRNA encoding hEPO or diluent (PBS). [Figure 4] Figure 4 is a graph showing that lipid A and lipid B LNP formulations (10 μg) with CA09 HA mRNA induced robust functional antibodies in cynomolgus macaques. HAI titers are reported as log2 for serum samples collected on study days 0, 14, 28, 42, and 56. [Figure 5A-1] Figures 5A-C show the MRT1400 mRNA encoding influenza virus A / Singapore / INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin. Figure 5A: Alignment of the wild-type (WT) gene and the codon-optimized gene (MRT10279) for the HA antigen. Figure 5B: mRNA structure. Figure 5C: mRNA sequence. [Figure 5A-2] Continued from Figure 5A-1. [Figure 5A-3] Continued from Figure 5A-2. [Figure 5A-4] Continuation of Figure 5A-3. [Figure 5A-5] Continuation of Figure 5A-4. [Figure 5B] Figures 5A-C show the MRT1400 mRNA encoding influenza virus A / Singapore / INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin. Figure 5A: Alignment of the wild-type (WT) gene and the codon-optimized gene (MRT10279) for the HA antigen. Figure 5B: mRNA structure. Figure 5C: mRNA sequence. [Figure 5C] Figures 5A-C show the MRT1400 mRNA encoding influenza virus A / Singapore / INFIMH160019 / 2016 (Sing16; H3N2) HA hemagglutinin. Figure 5A: Alignment of the wild-type (WT) gene and the codon-optimized gene (MRT10279) for the HA antigen. Figure 5B: mRNA structure. Figure 5C: mRNA sequence. [Figure 6] Figure 6 is a pair of graphs showing that lipid A and lipid B LNP formulations with MRT1400 or NA mRNA induced robust functional antibodies in mice. The first injection was given on study day 0, and the second injection was given on study day 28. Left panel: HAI titers are reported as log10 for serum samples collected on study days 14, 28, 42, and 56. Right panel: NAI titers are reported as log10 for serum samples collected on study days 14, 28, 42, and 56. Bars are geometric means and geometric standard deviations. Dashed line = lower limit of quantitation. [Figure 7A] Figure 7A is a graph showing that lipid A and lipid B LNP formulations with MRT1400 induced robust functional antibodies in NHPs. HAI titers are reported as log2 for serum samples collected on study days 0, 14, 28, 42, and 56. The first injection was given on study day 0, and the second injection was given on study day 28. Bars are the mean and standard deviation. Upper dashed line = 1 / 40 minimum target. Lower dashed line = lower limit of detection. [Figure 7B]Figures 7B and 7C are a pair of figures showing that a lipid A LNP formulation containing MRT1400 mRNA (MRT5400) induced functional antibodies (Figure 7B) and robust ELISA titers (Figure 7C) in cynomolgus 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 study days 0, 14, 28, 42, and 56. The first injection was given on study day 0, and the second injection was given on study day 28. Bars are 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 formulation containing MRT1400 mRNA (MRT5400) induced functional antibodies (Figure 7B) and robust ELISA titers (Figure 7C) in cynomolgus 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 study days 0, 14, 28, 42, and 56. The first injection was given on study day 0, and the second injection was given on study day 28. Bars are the mean and standard deviation. Dashed line = 1 / 40 minimum target. [Figure 8A] Figures 8A and 8B are graph panels showing T cell cytokine responses in cynomolgus macaques after a second vaccination with the lipid A LNP formulation 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 peptides (right panel) were assessed in peripheral blood mononuclear cells (PBMCs) on day 42 by ELISPOT assay. The frequency of PBMC-secreted IFN-γ (Figure 8A) or IL-13 (Figure 8B) was calculated as speck-forming cells (SFC) per million PBMCs. Each symbol represents an individual sample, and the bars represent the standard deviation. [Figure 8B]Figures 8A and 8B are graph panels showing T cell cytokine responses in cynomolgus macaques after a second vaccination with the lipid A LNP formulation 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 peptides (right panel) were assessed in peripheral blood mononuclear cells (PBMCs) on day 42 by ELISPOT assay. The frequency of PBMC-secreted IFN-γ (Figure 8A) or IL-13 (Figure 8B) was calculated as speck-forming cells (SFC) per million PBMCs. Each symbol represents an individual sample, and the bars represent the standard deviation. [Figure 9A] Figure 9A is a pair of graphs showing that lipid A LNP formulations containing modified and unmodified CA09 HA mRNA were comparable as indicated by HAI titers in vaccinated mice. HAI titers are reported as log2 for serum samples collected on study days 14, 28, 42, and 56. The first injection was given on study day 0, and the second injection was given on study day 28. Bars are the mean and standard deviation. Upper dashed line = 1 / 40 minimum target. Lower dashed line = lower limit of quantitation. [Figure 9B] Figure 9B is a pair of graphs showing that lipid A LNP formulations containing modified and unmodified CA09 HA mRNA were comparable in mice as indicated by ELISA titers. Total IgG ELISA titers are reported as log10 for serum samples collected on study days 14, 28, 42, and 56. The first injection was given on study day 0, and the second injection was given on study day 28. Dashed line = lower limit of quantitation. [Figure 10]Figures 10A and 10B are a pair of graphs showing that bivalent lipid A LNP formulations with CA09 HA mRNA and Sing16 HA mRNA induced robust functional antibodies, as assessed by HAI titers (CA09 (Figure 10A) and Sing16 (Figure 10B)), in Balb / c mice at a dose of 0.4 μg of total mRNA. 0.4 μg of mRNA was administered as a co-encapsulated mRNA-LNP formulation, or each HA mRNA was administered separately, with 0.2 μg delivered into each hind paw. Each HA mRNA was also co-encapsulated in the formulation with a non-coding mRNA to control for total mRNA loading in the LNP. Dilution groups received mRNA-LNP dilution buffer. HAI titers are reported for serum samples collected on study days -2 (baseline), 14, 28, and 42. Figure 10B shows only study days -2 (baseline from pooled serum) and 42. The first injection was given on study day 0 and the second injection was given on study day 28. Bars are geometric mean and geometric standard deviation. Dashed line = lower limit of quantitation. [Figure 11]Figure 11 shows functional validation of mRNA-LNP formulations. Panel (a) is a graph showing the expression of firefly (FF) luciferase in BALB / c mice: a single dose of luciferase FF mRNA-LNP (5, 1, 0.1, or 0.05 μg) was injected into mice (n = 4) via the IM route. Luciferin (3 mg) was injected during whole-body animal imaging using an IVIS Spectrum (Perkin Elmer) to record bioluminescence intensity. Images of whole-body animal mean luminescence were taken 6, 24, 48, and 72 hours after injection. The luminescence recorded for 1, 0.5, 0.1, and 0.05 μg doses of Luc mRNA-LNP are shown on the graph. Panel (b) shows whole-body animal images showing the total luminescence flux from 6 to 72 hours. The total luminescence flux for 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 by the IM route in BALB / c mice. hEPO expression was quantified in serum using ELISA at 6 and 24 hours after administration. Bars represent the mean and standard deviation. Panel (d) shows hEPO expression in NHPs. A single dose of hEPO mRNA-LNP (10 μg) was injected by the IM route in cynomolgus macaques. hEPO expression was quantified in serum using ELISA at 6, 24, 48, 72, and 96 hours after administration. Bars represent the mean and standard deviation. [Figure 12] Figure 12 shows serological evaluation of HA mRNA-LNP vaccines in mice. BALB / c mice (n = 8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of Cal09 HA mRNA-LNP or Sing16 HA mRNA-LNP. ELISA titers recorded for sera 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 serological evaluation of HA mRNA-LNP vaccines in mice. BALB / c mice (n = 8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of CA09 HA mRNA-LNP or Sing16 HA mRNA-LNP. Log HAI titers recorded against CA09 H1N1 influenza virus (left panel) and Sing16 H3N2 influenza virus (right panel) are shown. [Figure 14] Figure 14 shows serological evaluation of NA mRNA-LNP vaccines in mice. BALB / c mice (n = 8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP. Total IgG titers recorded for sera 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 serological evaluation of NA mRNA-LNP vaccines in mice. BALB / c mice (n=8 per group) were immunized IM twice, 4 weeks apart, with 2, 0.4, 0.08, and 0.016 μg of Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP. Log10 NAI (ELLA) titers recorded for sera 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 efficacy of the CA09 HA mRNA-LNP vaccine in mice after lethal A / Belgium / 2009 H1N1 virus challenge. Mice (n=8) received two IM doses of CA09 HA mRNA-LNP (0.4 μg each) on days 0 and 28. Control animals received two IM doses of diluent on days 0 and 28. Figure 16A shows HAI titers, reported as Log10, for serum samples collected on study days 0, 14, 28, 42, 56, 92, and 107. Figure 16B shows daily body weights after intranasal challenge with 4 LD50 of the A / Belgium / 2009 H1N1 strain on day 93. Body weights are presented as percentage weight loss from the day of challenge. Individual lines represent each animal. [Figure 17A] Figures 17A-B show the protective efficacy of a single dose of unmodified Mich15 NA mRNA-LNP in mice after a 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 (one dose) on test day 0, while the other half (two doses) received two injections given on test days 0 and 28. Control animals received two IM doses of hEPO mRNA-LNP (0.6 μg) on ​​days 0 and 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 daily body weight changes following intranasal challenge with 4LD50 of Belgium09 H1N1 on day 89 for the single-dose group and on day 117 for the two-dose group. Body weights are presented as percentage of weight loss from the day of challenge. Individual lines represent each animal. [Figure 17B]Figures 17A-B show the protective efficacy of a single dose of unmodified Mich15 NA mRNA-LNP in mice after a 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 (one dose) on test day 0, while the other half (two doses) received two injections given on test days 0 and 28. Control animals received two IM doses of hEPO mRNA-LNP (0.6 μg) on ​​days 0 and 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 daily body weight changes following intranasal challenge with 4LD50 of Belgium09 H1N1 on day 89 for the single-dose group and on day 117 for the two-dose group. Body weights are presented as percentage of weight loss from the day of challenge. Individual lines represent each animal. [Figure 18] Figure 18 shows serological evaluation of the HA Sing16 HA mRNA-LNP vaccine in NHPs. Cynomolgus macaques (n=6 per group) were injected twice, 4 weeks apart, with 15, 45, or 135 μg of Sing16 HA mRNA-LNPs via the IM route. Serum samples were collected on days -6, 14, 28, 42, and 56. Log10 IgG titers against the recombinant HA protein of the Sing16 virus are shown. [Figure 19] Figures 19A and 19B show serological evaluation of the HA Sing16 HA mRNA-LNP vaccine in NHPs. Cynomolgus macaques (n=6 per group) were injected twice, 4 weeks apart, with 15, 45, or 135 μg of Sing16 HA mRNA-LNP via the IM route. Serum samples were collected on days 0, 14, 28, 42, and 56. Log10 HAI titers (Figure 19A) and Log10 microneutralization (MN) titers (Figure 19B) against Sing16 virus are shown. [Figure 20]Figures 20A and 20B show T cell responses in NHPs vaccinated with the Sing16 HA mRNA-LNP vaccine. Cynomolgus macaques (n = 6 per group) were injected twice, 4 weeks apart, with 45, 135, or 250 μg of Sing16 HA mRNA-LNP via the IM route. T cells were assessed by ELISPOT on day 42 in PBMCs stimulated in vitro with a peptide pool representing the entire HA open reading frame. IFN-γ (Figure 20A) or IL-13 (Figure 20B) secreting PBMC responses, calculated as speck-forming cells (SFC) per million PBMCs, are shown. 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 on day 180 in NHPs vaccinated with the Sing16 HA mRNA-LNP vaccine. Cynomolgus macaques (n = 6 per group) were injected twice, 4 weeks apart, with 15 or 45 μg of Sing16 HA mRNA-LNP via the IM route. Sing16 / H3-specific and total IgG+ antibody-secreting cells (ASCs) were measured using a human IgG monochromatic memory B cell ELISPOT kit (CAT# NC1911372, CTL). Differentiation of MBCs into ASCs was performed on PBMCs collected on day 180 using the stimulation cocktail provided by the kit. The number of IgG+ and Sing16 / H3-specific ASCs was calculated per million PBMCs for each animal, and the frequency of antigen-specific ASCs is shown. [Figure 22-1]Figure 22 shows the delivery of bivalent influenza vaccine combinations in mice. BALB / c mice (n = 8 per group) were immunized twice, 4 weeks apart, IM with either 0.4 μg of the bivalent combination co-encapsulated mRNA transcripts (1:1 wt / wt, half a dose per leg) or 0.2 μg of each monovalent combination formulated separately and immunized in a different leg. The CA09 HA mRNA-LNP and Sing16 HA mRNA-LNP constituting the H1H3 combo; the Sing16 HA mRNA-LNP and Sing16 NA mRNA-LNP constituting the H3N2 combo; and the Mich15 NA mRNA-LNP and Perth09 NA mRNA-LNP constituting 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 against the Sing2016 H3N2 and A / Mallard / Sweden / 2002(H6) chimeric influenza viruses and the H6N2 A / Perth / 09 virus F1919D(N2) virus, respectively. Panel (c) shows the NAI titers recorded against the Mich15(N1):A / Mallard / Sweden / 2002(H6) chimeric influenza virus and the H6N2 A / Perth / 09 virus F1919D(N2) virus. [Figure 22-2] Continuation of Figure 22-1. [Figure 23]Figure 23 shows delivery of tetravalent combinations of influenza vaccines in NHPs. Cynomolgus macaques (n=6 per group) were immunized IM twice, 4 weeks apart, with 10 μg total of tetravalent combinations of co-encapsulated mRNA transcripts (1:1:1:1 wt / wt). 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 2x non-coding mRNA (ncmRNA); H3N2 combo of Sing16 HA mRNA, Perth09 NA mRNA, and 2x non-coding mRNA; N1N2 combo of Mich15 NA mRNA, Perth09 NA mRNA-LNP, and 2x non-coding mRNA; and H1 consisting of CA09 HA mRNA and 3x non-coding mRNA. H3 consists of Sing16 HA mRNA and 3x non-coding mRNA. N1 consists of Mich15 NA mRNA and 3x non-coding mRNA. N2 consists of Perth09 NA mRNA and 3x 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 depicts a graph showing human erythropoietin (hEPO) mRNA expression in mice treated with various LNP formulations of hEPO. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 25]Figure 25 depicts a graph showing hEPO expression in non-human primates (NHPs) treated with various LNP formulations of hEPO mRNA. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 26] Figure 26 depicts a graph showing HAI titers at 28 and 42 days after injection of various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 27] Figure 27 depicts a graph showing Cal09 H1 HAI titers at 28 and 42 days after injection of various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 28] Figure 28 depicts a graph showing Sing16 H3 HAI titers at 28 and 42 days after injection of various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid B," "Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent the mean and standard deviation. The LNP composition contains cationic lipid, DMG-PEG2000, cholesterol, and DOPE, in that order, in a molar ratio of 40:1.5:28.5:30. [Figure 29]Figure 29 shows the RSV F protein antibody titers in NHPs immunized with FD3 F protein-expressing mRNA. The mRNA was delivered in lipid nanoparticles (LNPs) containing one of several cationic lipids. Antibody titers were measured on days 0, 21, and 35 for each antigen composition. [Figure 30] Figure 30 depicts RSV neutralization titers in NHPs immunized with FD3 F protein-expressing mRNA. The mRNA was delivered in lipid nanoparticles (LNPs) containing one of several cationic lipids. Antibody titers were measured on days 0, 21, and 35 for each antigen composition. [Figure 31] FIG. 31 depicts the HAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to mice against four different influenza strains. [Figure 32] FIG. 32 depicts the HINT values ​​for tetravalent and octavalent mRNA-LNP vaccines administered to ferrets against four different influenza strains. [Figure 33] Figure 33 depicts the NAI titers for tetravalent and octavalent mRNA-LNP vaccines administered to mice against four different influenza strains. [Figure 34] Figure 34 depicts 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 vaccine. [Figure 35] Figure 35 depicts 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 vaccine. [Figure 36] Figure 36 depicts microneutralization titers for Sing16HA-encoding mRNA in lipid A LNP formulations administered to NHPs at 15 μg and 45 μg doses. Samples were obtained 6 days (D6) and 42 days (D42) after the second dose of vaccine. [Figure 37]Figure 37 depicts microneutralization titers for Sing16HA-encoding mRNA in lipid B LNP formulations administered to NHPs at 15 μg and 45 μg doses. Samples were obtained 6 days (D6) and 42 days (D42) after the second dose of vaccine. DETAILED DESCRIPTION OF THE INVENTION

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

[0035] The present inventors discovered this formulation through screening a combinatorial library of lipid components. The LNP encapsulates the mRNA payload, protecting it from degradation and promoting cellular uptake of the encapsulated mRNA. Compared to industrial formulations described in the literature, the LNP described herein enhances delivery efficiency and promotes endosomal escape of mRNA, 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) 28:172-6) 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):1570-8). As described further below, the present formulations encapsulating mRNA encoding hEPO, when delivered in vivo, resulted in high levels of circulating erythropoietin in the blood at 6 and 24 hours, up to a 12-fold increase compared to the industry standard, the MC3 LNP formulation. Similarly, high efficacy has been found with other mRNAs, such as those encoding influenza antigens, in both mouse and non-human primate models.

[0036] The mRNA vaccines formulated herein can be used to induce a balanced immune response, including both cellular and humoral immunity. The advantage of the LNP formulations is that they are not sequence-specific, allowing them to deliver mRNAs encoding a variety of antigens, enabling rapid deployment in epidemic or pandemic situations. Furthermore, the LNP-formulated mRNA vaccines are highly immunogenic, thus offering significant dose-sparing potential.

[0037] I. Composition of the Lipid Nanoparticles The present LNPs contain four categories of lipids: (i); ionizable lipids; (ii); PEGylated lipids; (iii) cholesterol-based lipids; and (iv) helper lipids.

[0038] A. Ionizable lipids Ionizable lipids facilitate mRNA encapsulation and may be cationic lipids, which provide a positively charged environment at low pH to facilitate 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 diketopiperazine core and a degradable ester bond for binding to a double unsaturated tail, while OF-02 contains the same diketopiperazine core and a non-degradable 1,2-amino alcohol bond for binding to a double unsaturated tail (Fenton et al., Adv Mater. (2016) 28: 2939; U.S. Patent No. 10,201,618). The exemplary LNP formulation herein, lipid A, contains OF-2.

[0041] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Patent No. 9,512,073).

[0042] [ka] An exemplary LNP formulation herein, lipid B, contains 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-hydroxyoctadec-9-en-1-yl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate, which is a HEPES-based disulfide cationic lipid with a piperazine core and has formula (III).

[0044] [ka] An exemplary LNP formulation 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 cationic lipid.

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

[0046] [ka] An exemplary LNP formulation 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 cationic lipid.

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

[0048] [ka] An exemplary LNP formulation 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 cationic lipid.

[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 presented in Scheme 1.

[0050] Scheme 1: General synthetic scheme for lipids of formula (III), (IV), and (V) [ka]

[0051] Other cationic lipids that can be used include those described in Dong, supra, and in US Pat. No. 10,201,618.

[0052] B. PEGylated lipids PEGylated lipid components control the particle size and stability of nanoparticles. The addition of such components can provide a means to prevent complex aggregation, increase circulation life, and enhance delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters (1990) 268(1):235-7). These components can be selected to rapidly clear the pharmaceutical composition in vivo (see, e.g., U.S. Patent No. 5,885,613).

[0053] Contemplated PEGylated lipids include C6-C ceramides, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (e.g., C8, C 10 , C 12 , C 14 , C 16 , or C 18 PEGylated lipids include, but are not limited to, polyethylene glycols (PEGs) up to 5 kDa in length covalently attached to a lipid having alkyl chain(s) of 1,2-dimethyl-3-methoxy-2-(2-methyl-2-methylpropyl)-2,3-dimethyl-2,4-dimethyl-2,5-dimethyl-2,6-dimethyl-2,7-dimethyl-2,8-dimethyl-2,9 ...

[0054] In particularly exemplary embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the pegylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000.

[0055] C. Cholesterol-based lipids The cholesterol component stabilizes the lipid bilayer structure inside the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-aryleneamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat. 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 The helper lipid enhances the structural stability of the LNP and aids in endosomal escape of the LNP. The helper lipid improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid, which has membrane fusogenic properties to enhance the uptake and release of the drug payload. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielideyl-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 are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations 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 the specific molar ratios of the above components are important for the effectiveness of LNPs in delivering mRNA. The molar ratio of cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid is A:B:C:D, where A+B+C+D=100%. In some embodiments, the molar ratio of cationic lipid in the LNP relative to total lipid (i.e., A) is 35-45% (e.g., 38-42%, such as 40%). In some embodiments, the molar ratio of the PEGylated lipid component relative to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, such as 1.5%). In some embodiments, the molar ratio of the cholesterol-based lipid relative to total lipid (i.e., C) is 20-35% (e.g., 27-30%, such as 28.5%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 25-35% (e.g., 28-32%, such as 30%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of cationic lipid to helper lipid of greater than 1.

[0060] In certain embodiments, the LNPs contain a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid in a molar ratio of 40:1.5:28.5:30. In further specific embodiments, the LNPs contain (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 molar ratio of 40:1.5:28.5:30.

[0061] To calculate the actual amount of each lipid 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 to be transported by the LNP. The molar amounts of each of the other lipids are then calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0062] F. Active ingredient of LNP The active ingredient of the LNP vaccine composition is mRNA encoding an antigen of interest, which 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), respiratory syncytial 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 respiratory syncytial virus (RSV).

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

[0064] If desired, LNPs or LNP formulations may be multivalent. In some embodiments, LNPs may carry 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, LNPs may carry multiple mRNA molecules, each encoding a different antigen; or may carry a polycistronic mRNA (e.g., each antigen-encoding sequence is separated by a nucleotide linker encoding a self-cleaving peptide, such as a 2A peptide) that can be translated into more than one antigen. LNPs carrying different mRNA molecules typically contain (encapsulate) multiple copies of each mRNA molecule. For example, LNPs carrying or encapsulating two different mRNA molecules typically carry multiple copies of each of the two different mRNA molecules.

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

[0066] An example of a multivalent 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 influenza virus. In some embodiments, the multivalent 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), nonstructural protein 1 (NS1), and nonstructural protein 2 (NS2). In further embodiments, the multivalent LNP vaccine contains two or more (e.g., three, four, five, six, seven, eight, or more) mRNA molecules encoding antigenic polypeptides derived from the HA protein, the NA protein, and both the HA and NA proteins. In some embodiments, the mRNA molecules encoding the antigenic polypeptides are from different influenza strains.

[0067] In certain embodiments, the composition may comprise one or more mRNA molecules encoding antigens of influenza A, B, and C viruses. In one embodiment, the composition may comprise 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 the 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 the 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 the influenza B virus are derived from the influenza B / Yamagata lineage. In one embodiment, the HA and NA antigens of the influenza B virus are derived from the influenza B / Victoria lineage. In some embodiments, one or more of the HA and NA antigens are derived from an influenza virus strain recommended by the World Health Organization (WHO) in its annual recommendations for influenza vaccine formulations.

[0068] In certain embodiments, at least one of the one or more influenza virus proteins comprises an influenza virus HA protein and / or an influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and at least one of the 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 two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding (i) one or more HA antigens, (ii) one or more NA antigens, or (iii) a combination of one or more HA and NA antigens.

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

[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 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 from the influenza B / Yamagata lineage, one mRNA molecule encoding an NA antigen from the influenza B / Yamagata lineage, one mRNA molecule encoding an HA antigen from the influenza B / Victoria lineage, and one mRNA molecule encoding an NA antigen from the influenza B / Victoria lineage.

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

[0074] Any machine learning algorithm may be used when selecting one or more machine learning influenza virus HAs, for example, any of the machine learning algorithms and methods disclosed in PCT Application WO 2021 / 080990, entitled SYSTEMS AND METHODS FOR DESIGNING VACCINES, and PCT Application WO 2021 / 080999, entitled SYSTEMS AND METHODS FOR PREDICTING BIOLOGICAL RESPONSES, both of which are incorporated by reference in their entireties.

[0075] mRNA molecules may be unmodified (i.e., contain only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or chemically modified (e.g., contain nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate linkages). mRNA molecules may include a 5' cap and a poly-A tail.

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

[0077] The RSV F protein is responsible for the fusion of the viral membrane with the host cell membrane and can assume at least three conformations: prefusion, intermediate, and postfusion. In the prefusion conformation (Pre-F), the F protein exists in a trimeric form, exposing the major antigenic site Φ. Site Φ serves as the primary target for neutralizing antibodies produced by RSV-infected subjects (see Coultas et al., Thorax. 74:986-993, 2019; McLellan et al., Science. 340(6136):1113-7, 2013). After binding to its target on the host cell surface, Pre-F undergoes a conformational change during which site Φ is no longer exposed. Pre-F then transitions to a transient intermediate conformation, allowing the F protein to insert into the host cell membrane, resulting in fusion of the viral and host cell membranes. A final conformational shift generates a more stable, elongated form of the protein (Post-F). Sites II and IV of the F protein are unique to Post-F, while site I is present in both the Pre-F and Post-F conformations (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 protein of RSV responsible for driving the fusion of the viral envelope with the host cell membrane during viral entry.

[0079] As used herein, the term "RSV F polypeptide" or "F polypeptide" refers to a polypeptide containing 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 conformation of RSV F that occurs after the combination of the viral and cellular membranes.

[0081] As used herein, the term "prefusion" with respect to RSV F refers to the conformation of RSV F that it adopts prior to virus-cell interaction.

[0082] Provided herein are mRNA molecules encoding antigenic RSV F polypeptides.

[0083] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) encoding a respiratory syncytial virus (RSV) F protein antigen.

[0084] In some embodiments, the RSV F protein antigen comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:16.

[0085] 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.

[0086] In some embodiments, the mRNA comprises 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 set forth in SEQ ID NO:17.

[0087] In some embodiments, the mRNA comprises 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 set forth in SEQ ID NO:21.

[0088] In some embodiments, the RSV F protein antigen is a prefusion protein.

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

[0090] In some embodiments, an 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.

[0091] In some embodiments, the mRNA comprises 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-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.

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

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

[0097] G. Buffers and Other Components To stabilize the nucleic acid and / or LNP (e.g., to extend the shelf life of a vaccine product), facilitate administration of the LNP pharmaceutical composition, and / or enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP 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 the like.

[0098] The LNP compositions of the present disclosure can be provided in a frozen liquid form or a lyophilized form. A variety of cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, at 5-30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP composition can be frozen (or lyophilized and stored frozen) at -20°C to -80°C.

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

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

[0101] II.A.5' Cap The mRNA 5' cap confers resistance to nucleases found in most eukaryotic cells and can promote translation efficiency. Several types of 5' caps are known: 7-methylguanosine cap ("m 7 Cap-0 (also called "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide through a 5'-5' triphosphate bond.

[0102] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, generating a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0103] 5'-Capping of polynucleotides may be completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs, which yield 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 modified RNAs may be completed post-transcriptionally using vaccinia virus capping enzyme, which generates the Cap 0 structure: m7G(5')ppp(5')G. Cap 1 structures may be generated using both vaccinia virus capping enzyme and a 2'-O-methyltransferase, which generates m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structures may be generated from the Cap 1 structure, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-O-methyltransferase. Cap 3 structures may be generated from the Cap 2 structure, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-O-methyltransferase.

[0104] In certain embodiments, an mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0105] In certain embodiments, the mRNA of the present disclosure comprises: [ka] Contains the 5' cap.

[0106] II.B. Untranslated Regions (UTRs) In some embodiments, the mRNA of the present disclosure comprises a 5' and / or 3' untranslated region (UTR). In the mRNA, the 5' UTR begins at the transcription initiation site and continues up to, but not including, the start codon. The 3' UTR begins immediately following the stop codon and continues to the transcription termination signal.

[0107] In some embodiments, the mRNAs disclosed herein may comprise a 5' UTR that contains one or more elements that affect mRNA stability or translation. In some embodiments, the 5' UTR may be about 10-5,000 nucleotides in length. In some embodiments, the 5' UTR may be about 50-500 nucleotides in length. In some embodiments, the 5' UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 800 nucleotides in length, about 900 nucleotides in length, about 1000 nucleotides in length, about 1500 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 800 nucleotides in length, about 900 nucleotides in length, about 1000 nucleotides in length, about 1000 nucleotides in length, about 1500 nucleotide 0 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or 5,000 nucleotides in length.

[0108] In some embodiments, the mRNAs disclosed herein may include a 3' UTR that includes one or more polyadenylation signals, binding sites for proteins that affect the stability of the mRNA's location within a cell, or one or more binding sites for miRNAs. In some embodiments, the 3' UTR may be about 50-5,000 nucleotides in length or longer. In some embodiments, the 3' UTR may be about 50-1,000 nucleotides in length or longer. In some embodiments, the 3'UTR is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0109] In some embodiments, the mRNAs disclosed herein may include a 5' or 3' UTR that is derived from a gene that is different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

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

[0111] Exemplary 5'UTRs include sequences from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO: 22) (U.S. Patent Application Publication No. 2016 / 0151409, which is 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 a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication No. 2017 / 0029847, U.S. Patent Application Publication No. 2016 / 0304883, U.S. Patent Application Publication No. 2016 / 0235864, and U.S. Patent Application Publication No. 2016 / 0166710, each of which is incorporated herein by reference).

[0113] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, see above).

[0114] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, see above).

[0115] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, see above).

[0116] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

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

[0118] II.C. Polyadenylation Tail As used herein, the terms "poly(A) sequence," "poly(A) tail," and "poly(A) region" refer to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have a length of essentially 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide other than adenosine nucleotides (e.g., a nucleotide that is not adenosine nucleotides). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide other than adenosine nucleotides, or a stretch of nucleotides). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 23).

[0119] "Poly(A) tail," as used herein, typically relates to RNA. However, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0120] The poly(A) tail may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[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 in vitro transcription of RNA. In certain embodiments, the poly(A) tail is obtained by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of RNA using a commercially available polyadenylation kit and corresponding protocol, or alternatively, by using immobilized poly(A) polymerase, for example, by using the methods and means described in WO2016 / 174271.

[0122] The nucleic acid may contain a poly(A) tail obtained by enzymatic polyadenylation, with the majority of nucleic acid molecules containing from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0123] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from the template DNA, and may further comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO2016 / 091391, which is incorporated herein by reference.

[0124] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

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

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

[0127] II.D. Chemical Modification The mRNAs disclosed herein may be modified or unmodified. In some embodiments, the mRNA may include at least one chemical modification. In some embodiments, the mRNAs disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNAs may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNAs may be selected from a group consisting of, 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-bromo ... and modified nucleotide analogs or derivatives of purines and pyrimidines such as 5-mo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouracil, queusine, β-D-mannosyl-queusine, phosphoramidate, phosphorothioate, peptide nucleotides, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0128] In some embodiments, the disclosed mRNAs may contain at least one chemical modification including, but not limited to, 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.

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

[0130] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0131] 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.

[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 preparation of such analogs is described, for example, in U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642.

[0134] II.E. mRNA Synthesis The mRNA disclosed herein may be synthesized according to any of a variety of methods. For example, mRNA according to the present disclosure may be synthesized by in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions may vary according to 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 can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0135] III. Methods for Making the Present LNP Vaccine The LNPs can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by dissolving lipids in a suitable solvent and then evaporating the solvent to leave a thin film on the inside of a vessel, or by spray-drying to deposit selected lipids on the inner walls of a suitable container or vessel. An aqueous phase can then be added to a vortexing vessel to form MLVs. Unilamellar liposomes (ULVs) can then be formed by homogenizing, sonicating, or extruding the multilamellar vesicles. Additionally, unilamellar 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 practice the present invention. One exemplary method involves encapsulating mRNA by combining the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in US2016 / 0038432. Another exemplary method involves encapsulating mRNA by combining preformed LNPs with the mRNA, as described in US2018 / 0153822.

[0137] In some embodiments, a method for producing mRNA-loaded LNPs includes heating one or more solutions to a temperature above ambient temperature, the one or more solutions being a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing LNP-encapsulated mRNA. In some embodiments, the method includes heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step. In some embodiments, the method includes heating one or more of the solution containing preformed LNPs, the solution containing mRNA, and the solution containing LNP-encapsulated mRNA during the mixing step. In some embodiments, the method includes heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions is 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 to which one or more of the solutions are heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature is about 65° C.

[0138] Various methods may be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA may be directly dissolved in the buffer solution described herein. In some embodiments, mRNA solutions may be prepared by mixing mRNA stock solutions with buffer solutions before mixing with lipid solutions for encapsulation. In some embodiments, mRNA solutions may be prepared by mixing mRNA stock solutions with buffer solutions immediately before mixing with lipid solutions for encapsulation. In some embodiments, suitable mRNA stock solutions may contain mRNA in water or buffer at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 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 at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x that of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min, or greater.

[0140] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or greater.

[0141] The method of incorporating desired mRNA into lipid nanoparticles is called "loading." Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The LNP-incorporated nucleic acid may be located completely or partially in the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the outer surface of the lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as "encapsulation," in which the nucleic acid is completely or substantially contained within the internal space of the lipid nanoparticle.

[0142] Suitable LNP can be produced in various sizes.In some embodiments, the size reduction of lipid nanoparticles is associated with more efficient delivery of mRNA.Selection of suitable LNP size can take into account the location of target cell or tissue and to some extent the application that lipid nanoparticles are produced for.

[0143] A wide variety of methods known in the art are available for sizing a population of lipid nanoparticles. A particularly exemplary method herein utilizes a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed in the Zetasizer instrument. The z-average diameter (nm), or cumulant average, is considered to be the average size for the LNPs in the sample. The Zetasizer instrument can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonicating the formed LNPs. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) evaluation to guide efficient lipid nanoparticle synthesis.

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

[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, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 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 via inhalation administration.

[0147] In some embodiments, the dispersity, or molecular size heterogeneity measure (PDI), of the LNPs in the pharmaceutical compositions provided herein is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI may be measured by a Zetasizer machine, as described above.

[0148] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 90%, or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).

[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 of greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0150] In some embodiments, pharmaceutical compositions according to the present invention contain at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, pharmaceutical compositions contain 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, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding a cap and a tail, and the modified RNA is again purified by depth filtration and TFF.

[0152] The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer can 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 producing mRNA-LNP compositions is described in Example 1. The method involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled, homogeneous manner, with the lipid-to-mRNA ratio maintained throughout the mixing process. In this example, mRNA is presented in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanolic lipid solution are mixed at a 4:1 volume ratio in a "T" mixer with an approximately "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. TFF may be used to concentrate and buffer exchange the nascent LNPs obtained immediately after formation by the T-mixing method. The diafiltration process is a continuous operation, and the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow.

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

[0155] Thus, the present disclosure provides kit-like products that provide an mRNA-LNP vaccine in a single container, or provide an mRNA-LNP vaccine in one container and a physiological buffer for reconstitution in another container. The container(s) may contain single-use or multi-use dosages. The containers may be pre-processed glass vials or ampoules. The products may also include instructions for use.

[0156] In certain embodiments, the mRNA-LNP vaccine is provided for use in intramuscular (IM) injection. The vaccine can be injected into the deltoid muscle of the subject's upper arm, for example. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for use in inhalation, and is provided in a pre-filled pump, intratracheal nebulizer, or inhaler.

[0157] The mRNA-LNP vaccine is administered to a subject in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against the target pathogen for a sufficient time (e.g., 1 year, 2 years, 5 years, 10 years, or lifelong). Sufficient immune protection may, for example, be the prevention or alleviation of symptoms associated with infection by the pathogen. In some embodiments, multiple doses (e.g., two doses) of the vaccine are injected into a subject in need thereof to achieve the desired preventive effect. Administration (e.g., prime and booster administrations) 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 of mRNA-LNP vaccine contains 1-50 μg of mRNA (e.g., monovalent or multivalent). For example, a single dose may contain about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg of mRNA for intramuscular (IM) injection. In further embodiments, a multivalent single dose of LNP vaccine contains multiple (e.g., 2, 3, or 4) types of LNPs, each for a different antigen, with each type of LNP having, for example, an mRNA amount of about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg.

[0159] In another aspect, the present invention provides methods of immunizing a subject against one or more influenza viruses. The present invention further provides methods of inducing an immune response in a subject against one or more influenza viruses. In some embodiments, the method comprises administering to the subject an effective amount of a composition described herein.

[0160] In various embodiments, the immunization methods provided herein induce a broadly protective immune response against multiple epitopes within one or more influenza viruses. In various embodiments, the immunization methods provided herein induce a broadly neutralizing immune response against one or more influenza viruses. In some embodiments, the immune response comprises an antibody response. Thus, in various embodiments, the compositions described herein can confer broad cross-protection against different types of influenza viruses. In some embodiments, the compositions confer cross-protection against avian, swine, seasonal, and / or pandemic influenza viruses. In some embodiments, the compositions confer cross-protection against one or more influenza A, B, or C subtypes. In some embodiments, the compositions confer cross-protection against multiple strains of influenza A H1 subtype viruses (e.g., H1N1), influenza A H3 subtype viruses (e.g., H3N2), influenza A H5 subtype viruses (e.g., H5N1), and / or influenza B viruses (e.g., Yamagata lineage, Victoria lineage).

[0161] In some embodiments, the methods of the present invention can elicit 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, A / Solomon Islands / 03 / 2006, A / Brisbane / 59 / 2007, A(H3N2) viruses antigenically similar to the cell-transmitted prototype virus A / Victoria / 361 / 2011, A / Beijing / 262 / 95(H1N1)-like viruses, A / Brisbane / 02 / 2018(H1N1)pdm09-like viruses, A / Brisbane / 10 / 2007(H3N2)-like viruses, A / California / 7 / 2004(H3N2)-like viruses, A / California / 7 / 2009(H1 N1)-like virus, A / California / 7 / 2009(H1N1)pdm09-like virus, A / Cambodia / e0826360 / 2020(H3N2)-like virus, A / Fujian / 411 / 2002(H3N2)-like virus, A / Fujian / 411 / 2002(H3N2)-like virus, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09-like virus, A / Hawaii / 70 / 2019(H1N1)pdm09-like virus, A / Hong Kong Kong / 2671 / 2019(H3N2)-like virus, A / Hong Kong / 45 / 2019(H3N2)-like virus, A / Hong Kong / 4801 / 2014(H3N2)-like virus, A / Kansas / 14 / 2017(H3N2)-like virus, A / Michigan / 45 / 2015(H1N1)pdm09-like virus, A / Moscow / 10 / 99(H3N2)-like virus, A / New Caledonia / 20 / 99(H1N1)-like virus, A / Perth / 16 / 2009(H3N2)-like virus, A / Singapore / INFIMH-16-0019 / 2016(H3N2)-like virus, A / SolomonIslands / 3 / 2006(H1N1)-like virus, A / South Australia / 34 / 2019(H3N2)-like virus, A / Switzerland / 8060 / 2017(H3N2)-like virus, A / Switzerland / 9715293 / 2013(H3N2)-like virus, A / Sydney / 5 / 97(H3N2)-like virus, A / Texas / 50 / 2012(H3N2)-like virus, A / Victoria / 2570 / 2019(H1N1)pdm09-like virus, A / Victoria / 2570 / 2019(H1N1)pdm09-like virus, A / Victoria / 361 / 2011(H3N2)-like virus Rus, A / Wellington / 1 / 2004(H3N2)-like virus, A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Wisconsin / 67 / 2005(H3N2)-like virus, B / Beijing / 184 / 93-like virus, B / Brisbane / 60 / 2008-like virus, B / Colorado / 06 / 2017-like virus (B / Victoria / 2 / 87 lineage), B / Florida / 4 / 2006-like virus, B / Hong Kong-like virusIncluding, but not limited to, Kong / 330 / 2001-like viruses, B / Malaysia / 2506 / 2004-like viruses, B / Massachusetts / 2 / 2012-like viruses, B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like viruses, B / Phuket / 3073 / 2013-like viruses, B / Phuket / 3073 / 2013-like viruses (B / Yamagata / 16 / 88 lineage), B / Shangdong / 7 / 97-like viruses, B / Shanghai / 361 / 2002-like viruses, B / Sichuan / 379 / 99-like viruses, B / Washington / 02 / 2019 (B / Victoria lineage)-like viruses, B / Washington / 02 / 2019-like (B / Victoria lineage) viruses, and B / Wisconsin / 1 / 2010-like viruses. In some embodiments, the methods of the present invention can elicit 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, among others, H1N1, H5N1, H2N2, H3N2, H9N2, H7N7, H7N3, H7N9, and H10N7 subtypes. In some embodiments, the methods of the present invention can elicit an improved immune response against one or more swine influenza strains. Exemplary swine strains include, but are not limited to, the A / New Jersey / 1976 isolate and A / California / 07 / 2009. In some embodiments, the methods of the present invention can elicit an improved immune response against 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 swine strains are known in the art.

[0162] In some embodiments, the present invention provides methods 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 has or is susceptible to influenza infection. In some embodiments, a subject is considered to have influenza infection if the subject exhibits one or more symptoms commonly associated with influenza infection. In some embodiments, a subject is known or believed to have been exposed to an influenza virus. In some embodiments, a subject is considered susceptible to influenza infection if the subject is known or believed to have been exposed to an influenza virus. In some embodiments, a subject is known or believed to have been exposed to an influenza virus if the subject has been in contact with another individual known or suspected to be infected with the influenza virus and / or if the subject is in or has been in a location where influenza infection is known or believed to be prevalent.

[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 a prophylactic. In such embodiments, the methods of the invention are effective in preventing or protecting a subject from influenza virus infection. In some embodiments, the compositions of the invention are used as a component of a seasonal and / or pandemic influenza vaccine or as part of an influenza vaccination regimen intended to provide long-lasting (multi-season) protection. In some embodiments, the compositions of the 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 a livestock or pet (e.g., a dog, cat, sheep, cow, and / or pig). In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a bird (e.g., a chicken, duck, goose, and / or turkey).

[0165] In some embodiments, the subject is a human. In certain embodiments, the subject is an adult, an adolescent, or an infant. In some embodiments, the human subject is younger than 6 months of age. In some embodiments, the human subject is 6 months or older, 6 months to 35 months, 36 months to 8 years, or 9 years or older. In some embodiments, the human subject is 60 years or older, or 55 years or older, such as 65 years or older. Administration of the compositions and / or performance of the treatment methods in utero is also contemplated herein.

[0166] Unless otherwise defined herein, scientific and technical terms used in connection with this specification shall have the same meaning as commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In the case of conflict, the present specification, including definitions, will control. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include plurals, and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the terms "approximately" or "about," as applied to one or more values ​​of interest, refer to values ​​that are similar to the stated reference value. In certain embodiments, the 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 (upper or lower) of the stated reference value, unless otherwise stated or apparent from the context.

[0167] In order that this invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. [Example]

[0168] Optimization of LNP formulation This example describes a study in which a series of LNP formulations for mRNA vaccines were produced from a combinatorial library of various components. Rationally designed novel cationic lipids were synthesized. In total, over 150 lipids and over 430 formulations were tested. Human erythropoietin (hEPO) mRNA was used as the test mRNA. In the primary formulation described below, mRNA is formulated into LNPs using various permutations of cationic lipids in combination with three other lipids: helper lipids; cholesterol-based lipids; and PEGylated lipids.

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

[0170] Citrate buffer (1 mM citric acid, 150 mM NaCl, pH 4.5) was used to prepare 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 the lipid ethanol solution and mRNA citrate solution in a "T" mixer using a pump system. The resulting solution then underwent buffer exchange using TFF / dialysis tubing. The concentration of the final formulation in 10% (w / v) trehalose was adjusted based on dosing requirements.

[0172] In vivo expression of hEPO protein in mice was used as a surrogate for measuring the efficacy of LNPs to deliver mRNA in vivo. In this study, a single dose (0.1 μg) of hEPO mRNA formulated in LNPs from various combinations of components was injected intramuscularly (IM) into mice. Serum collected 6 and 24 hours after administration was tested for hEPO levels using ELISA. The industry benchmark MC3 formulation was used as a reference for calculating the fold increase in hEPO expression (Angew, Chem Int Ed. (2012) 51:8529-33).

[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 robust efficacy compared to the MC3 formulation.

[0174] Further formulations were tested. The optimized formulations, Lipid A LNP and Lipid B LNP, are shown in Table 1. The mRNA in these formulations can be modified or unmodified and can encode antigens from viruses such as influenza or SARS-CoV-2.

[0175] [Table 1]

[0176] In Table 1, the final dose for a human vaccine is considered to be a dilution of the above 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, converted to the anhydrous component using the ratio of the molecular weight values ​​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 fold increase in hEPO expression with lipid A and lipid B compared to MC3 indicates that these LNPs are superior to MC3 in delivering mRNA. In the table below, "P2" means PEG2000; "Times MC3" means fold increase relative to MC3; and "Std Dev" means standard deviation.

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

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

[0182] One of the key process parameters identified during optimization was the flow rate during the initial blending step. Formulations with different final LNP sizes (ranging from 108 to 177 nm) were produced by varying these flow rates during blending, allowing additional control over process and product attributes. The higher the flow rate, the smaller the particle size. Potency increased significantly when the flow rate reached 375 ml / min, producing an average LNP size of 108 nM. The effect of size on LNP potency was noted as a measure of fold increase in hEPO expression relative to MC3 in Table 4.

[0183] [Table 4]

[0184] The above screening data indicate that the helper lipid DOPE was effective in promoting protein expression. The data also led to the determination of a promising molar composition 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 demonstrated desirable characteristics and freeze / thaw cycle stability. The long-term stability of the formulations was evaluated at -80°C in 10% (w / v) trehalose. Lipid A and Lipid B formulations were found to be highly stable. [Example]

[0185] Influenza HIN1 LNP vaccine formulation Influenza pandemics can occur when a novel influenza virus emerges in the human population. Such pandemics remain a major threat to public health, requiring vigilance and preparation with protective measures to be used in the event of prolonged human-to-human transmission of the virus. In the experiments described in this example, the hemagglutinin (HA) from the highly pathogenic HIN1 strain A / California / 7 / 2009 (CA09), which was responsible for the 2009 influenza pandemic, was used as a prototype antigen to evaluate the efficacy of mRNA vaccines produced with lipid A and lipid B LNP formulations.

[0186] HA mRNA was produced as described above. Citrate buffer (1 mM citrate, 150 mM NaCl, pH 4.5) was used to prepare the LNP composition. The citrate buffer containing the mRNA was mixed with lipids in 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 homogenous, pulseless flow, and the lipids and mRNA were mixed at a constant ratio throughout the process. This was crucial for achieving a homogenous formulation with the desired size and a low PDI, an indicator of a more homogenous size distribution. This method resulted in high mRNA encapsulation, which is crucial for achieving high efficacy. The resulting solution then underwent buffer exchange using TFF / dialysis tubing.

[0187] In a mouse study, the efficacy of lipid A and lipid B CA09 HA formulations was evaluated in a head-to-head comparison with the MC3 LNP formulation and recombinant HA (rHA). Balb / C mice (n = 8) were injected intramuscularly with CA09(H1) HA mRNA (0.4 μg) formulated with different cationic lipids on days 0 (DO) and 28 (D28). Vaccine immunogenicity, as indicated by HA inhibition (HAI) titers, is shown in Figure 2A. The data show that dual immunization with lipid A or lipid B on days 0 (DO) and 28 (D28) induced high HAI titers and completely protected the animals from a homologous virus challenge (Belgium09 H1N1 virus) (Figure 2B). During the 14-day post-challenge observation period, no overt signs of morbidity (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 showed morbidity (Figure 2B).

[0188] Similarly, mRNA encoding neuraminidase (NA) from the Mich15 influenza strain (Mich15 N1) was formulated with lipid A and its efficacy was evaluated. Balb / c mice (n = 8) were intramuscularly injected with two doses (0.4 or 0.016 μg) of NA mRNA formulated with lipid A. A control group (n = 8) was injected with 0.6 μg of hEPO mRNA or diluent. Half of the mice received only one injection (1 dose) on study day 0, while the other half received two injections (2 doses) given on study days 0 and 28. The data show that this N1 lipid A formulation induced a robust immune response, as indicated by NA inhibition (NAI) titers (Figure 3A). The data further show that mice treated with one or two doses of vaccine were protected from a lethal viral challenge with Belgium09 H1N1 (Figure 3B). The level of protection correlated with the NAI titers in the vaccine-treated groups versus the negative control groups (hEPO and diluent).

[0189] CA09 H1 mRNA formulated with the present LNPs was also tested in the NHP model. mRNA (10 μg) was formulated with lipid A and lipid B and injected intramuscularly into cynomolgus macaques (n=6) on study days 0 and 28. Detectable HAI priming by day 14 and a significant boost in HAI titers by day 28 were observed for all LNPs (Figure 4, right panel). ELISA data also showed significant priming above baseline by day 14 for all doses tested, with a robust boost detected 2 weeks post-boost (Figure 4, left panel). The results demonstrate that the present H1 mRNA formulation generated a robust immune response, as indicated by HAI and endpoint ELISA titers. [Example]

[0190] Influenza H3N2 LNP vaccine formulation This example describes experiments in which mRNA-LNP vaccine formulations against influenza strain Sing16 (H3N2) were evaluated for efficacy. 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 sequence of influenza virus hemagglutinin, H3 subtype, is shown below. MKTIIALSYI LCLVFAQKIP GNDNSTATLC LGHHAVPNGT IVKTITNDRI EVTNATELVQ NSSIGEICDS PHQILDGENC TLIDALLGDP QCDGFQNKKW DLFVERSKAY SNCYPYDVPD YASLRSLVAS SGTLEFKNES FNWTGVTQNG TSSACIRGSS SSFFSRLNWL THLNYTYPAL NVTMPNKEQF DKLYIWGVHH PGTDKDQIFL YAQSSGRITV 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* (SEQ ID NO: 1)

[0192] The coding sequence for this protein was codon-optimized. The codon-optimized sequence encoding 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 is flanked by 5' and 3' untranslated regions (UTRs) of 140 and 100 nucleotides, respectively. The biosynthetic HA-H3 mRNA contains a 7-methylguanosine (mGU) residue linked to the first nucleoside of the 5'UTR via an inverted 5'-5' triphosphate bridge. 7The HA-H3 mRNA also contains a 5'-cap structure consisting of a 5'-G residue, the first nucleoside of which is itself modified by 2'-O-ribose methylation. The 5'-cap is essential for ribosomal initiation of translation. The entire linear structure is terminated at the 3' end by a tract of approximately 100-500 adenosine nucleosides (polyA). The polyA region is also thought to confer stability to the mRNA and enhance translation. All of these structural elements are naturally occurring components used to promote efficient translation of the HA-H3 mRNA.

[0193] A DNA plasmid was constructed for producing codon-optimized mRNA sequences by in vitro transcription. In vitro transcription (IVT) reactions were performed using RNA polymerase. The reaction mixture was precipitated. The precipitated RNA samples were loaded onto individual depth filtration cassettes, washed with 80% ethanol, and redissolved in recycled water. A second aliquot of water was pumped out in a manner similar to the first step. This step was repeated once more. The pooled eluate underwent ultrafiltration / diafiltration using a 50 kD hollow fiber TFF cassette. Each IVT TFF pool was then diluted in preparation for the cap and tail reactions. The cap-tail reactions were precipitated, and the RNA from the reactions 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 with lipid A or lipid B and injected intramuscularly into Balb / c mice (n = 8) at 0.4 μg of mRNA per dose 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) 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 elicited robust HAI responses after a boost on day 28 (Figure 6, left panel).

[0196] Similarly, Sing16 HA mRNA lipid A and lipid B vaccines were evaluated in non-human primate (NHP) cynomolgus macaques (n=6). HA Sing16 mRNA (50 μg) formulated with lipid A or lipid B was injected intramuscularly into the monkeys. 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 functional response that was boosted on day 28 (Figure 7A).

[0197] Additionally, four dose levels of HA Sing16 mRNA formulated with lipid A (i.e., MRT5400 vaccine) were evaluated in NHPs: 15, 45, 135, and 250 μg. The first immunization was given on study day 0, and the second immunization on study day 28. All NHPs demonstrated IgG binding and HAI titers for all doses tested, with no differences in immune responses 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 responses in NHPs after the second vaccination. Peripheral blood mononuclear cells (PBMCs) were collected on day 42 and incubated overnight with Sing16 H3 recombinant protein or a peptide pool representing the entire HA open reading frame. Cytokines induced by restimulation were assessed by ELISPOT assay. The frequency of PBMCs secreting IFN-γ, a Th1 cytokine (Figure 8A), or IL-13, a Th2 cytokine (Figure 8B), was calculated as spot-forming cells (SFC) per million PBMCs. The majority of animals in the three dose-level groups tested (250 μg, 135 μg, and 45 μg) showed a high frequency of IFN-γ-secreting cells, exceeding 100 SFC per million PBMCs (Figure 8A). No dose response was observed, as animals in the lower and higher dose groups 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 groups tested at any dose level (Figure 8B). These data provide clear evidence of a Th1-biased cellular response and a lack of Th2 responses to HA antigen following vaccination in NHPs. [Example]

[0199] Influenza LNP vaccine formulations with modified mRNA This example describes an experiment comparing the efficacy of vaccines containing unmodified (unmodified non-replicating or "UNR") and modified (modified non-replicating or "MNR") mRNA. UNR CA09 HA mRNA and MNR CA09 HA mRNA were produced by in vitro transcription. In MNR, all uridines were replaced with pseudouridines.

[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 intramuscularly into Balb / c mice (n = 15). The data indicate that the LNP formulation increased the stability and delivery efficiency of naked mRNA (UNR), as the potency between UNR and MNR mRNA was comparable as indicated by HAI titers (Figure 9A). ELISA data for Balb / c mice also showed significant priming above baseline by day 14 for all doses tested (both UNR and MNR mRNA), with a robust boost detected 2 weeks post-boost. The data also indicate that UNR and MNR mRNA were comparable in eliciting ELISA titers (Figure 9B).

[0201] In conclusion, this dose titration study demonstrated that unmodified and modified CA09 HA mRNA formulated with lipid A elicited statistically indistinguishable immune responses in Balb / c mice, as demonstrated by HAI or endpoint ELISA assays. Balb / c mice immunized with the four higher doses of UNR and MNR mRNA exhibit detectable HAI priming by day 14 and a significant boost in HAI titers by day 42 for all doses. These day 14 priming titers represent both a dose effect and potential dose sparing, producing detectable titers over a 125-fold range. Secondary injection titers over the same dose range confirm the robustness of the immune response to this mRNA-LNP formulation. Similar results were observed in nonhuman primates. [Example]

[0202] Multivalent influenza vaccine LNP formulation 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, lipid A-coencapsulated CA09 HA mRNA and Sing16 HA mRNA were evaluated in Balb / c mice (n=8). mRNA-LNPs were administered as two coencapsulated mRNAs or separately as a single encapsulated mRNA. For both approaches, mice were injected intramuscularly with a total of 0.4 μg of LNP formulation. 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 elicited a robust immune functional response. There did not appear to be any differences between the two administration approaches. These data indicate that coencapsulation did not cause any interference or disruption between the two mRNAs. [Example]

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

[0205] material and method mRNA-LNP formulation 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 resulting purified mRNA precursors were further reacted by enzymatically adding a 5' cap structure (Cap1) and an approximately 200-nucleotide-long 3' poly(A) tail, determined by gel electrophoresis and purified. All mRNA preparations were analyzed for purity, integrity, and percentage of Cap1 before storage at -20°C. The preparation of mRNA / lipid nanoparticle (LNP) formulations was described above. Briefly, an ethanolic solution of a lipid mixture (ionizable lipid, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipid) with a fixed lipid-to-mRNA ratio was combined with an aqueous buffer solution of target mRNA at acidic pH under controlled conditions to obtain a homogeneous LNP suspension. The resulting nanoparticle suspension was diluted to the final concentration by ultrafiltration and diafiltration into an appropriate diluent system, filtered, and stored frozen at -80°C until use. The mRNA-LNP formulations were characterized for size by dynamic light scattering, percentage encapsulation, and stored at -80°C at 1 mg / mL upon dilution with an appropriate buffer until further use. hEPO-LNPs and FF-LNPs were utilized to examine the level 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 LNPs using a previously described method (Kalnin et al., npj Vaccines (2021) 6:61). Cells were fixed in 4% paraformaldehyde and subjected to targeted antibody staining for HA (GeneTex GTX40258), NA, and the ER marker Calnexin (Abcam ab22595). Images were captured on a confocal microscope and subsequently analyzed for quantification of HA and NA colocalization to the ER, mean signal intensity, and percent cell area.

[0207] Flow cytometry Human skeletal muscle cells (HskMCs, Lonza) were cultured in M199 (Life Technologies) supplemented with GlutaMAX (Life Technologies), streptomycin, penicillin (Gibco), and 20% heat-inactivated FBS (VWR) at 37°C and 5% CO. Cells were harvested by trypsinization, washed with PBS, and electroporated for 10 min according to the manufacturer's electroporation program D-033. 6 Twelve milligrams of mRNA per cell was electroporated using a human primary muscle cell transfection kit on a Nucleofector 2b (Lonza) transfection system. After 24 hours, harvested cells were fixed and permeabilized with Cytofix™ / Perm (BD) and 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 a PE-conjugated goat anti-mouse IgG secondary antibody (Southern Biotech) or an AF647-conjugated goat anti-rabbit IgG (Life Technologies). Antibody-labeled cells were then acquired using a Fortessa (BD) transfection system, and the expression of each protein was analyzed using FlowJo™ (TreeStar).

[0208] cryo-transmission electron microscope Grids were plasma-cleaned using a PELCO easiGlow™ device prior to LNP sample application, and a Vitrobot Mark IV system (ThermoFisher) with a chamber maintained at 100% humidity and 18°C ​​was used for plunge freezing. A 3.0 μl droplet of LNP sample was 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, and then immediately plunge-frozen into 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) operated in counting mode. The calibrated physical pixel size on the detector was 1.38 Å, corresponding to a magnification of 64,000. A total of 3,141 69-frame movie exposures were collected with a defocus of -0.5 to -1.7 μm and a per-frame dose of 1.045 e / Å. For each movie exposure, patch-based motion correction, super-resolution pixel binning, and frame weighting were performed using RELION-3.1.34. Over 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 NHPs for expression studies Groups of four cynomolgus macaques (NHPs) (male and female) and four to eight male BALB / c mice were intramuscularly administered either 10 μg (NHPs) or 1, 0.5, 0.1, and 0.05 μg (mice) of hEPO-LNPs manufactured at the same ratio intended for the HA / NA mRNA-LNP formulation. Blood samples were collected pre-dose and 6, 24, 48, 72, and 96 h post-dose and monitored for serum hEPO expression by ELISA using R&D Systems, Quantikine® IVD® ELISA, and human erythropoietin immunoassay kits according to the manufacturer's protocol. Results were reported as final values ​​in mIU / ml and ng / ml. Briefly, microplate wells precoated with a mouse monoclonal antibody specific for EPO were incubated with specimens or standards. After removing excess sample or standard, the wells were incubated with a 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 chromogen was added to the wells, and the chromogen was oxidized by the enzymatic reaction to form a blue complex. The reaction was stopped by adding acid, which changed the blue color to yellow. The amount of color produced was directly proportional to the amount of conjugate bound to the EPO-antibody complex, which in turn was directly proportional to the amount of EPO in the sample or standard. The absorbance of this complex was measured, and a standard curve was constructed by plotting absorbance versus the concentration of the EPO standard. The EPO concentration of unknown samples was determined by comparing the optical density of the sample to the standard curve. The standard used in this assay was recombinant hEPO, a urinary form of human erythropoietin, calibrated against a second international reference standard (67 / 343).

[0210] Immunization of mice and NHPs for immunogenicity studies Groups of Balb / c mice (Mus musculus) according to treatment group were immunized under isoflurane anesthesia in the quadriceps by the IM route in one hind limb on day 0 and the contralateral limb on day 28 with a dose of 0.05 mL of the designated vaccine formulation or diluent. Mice that lost more than 20% of their initial body weight and showed significant clinical signs were euthanized prior to study termination after veterinary evaluation of the animal's health.

[0211] Naive male and female crab-eater macaques (Macaca fascicularis) originating from Mauritius were selected for this study. At the start of the study, animals weighed >2 kg and were >2 years old. Animals selected for the study underwent an extensive physical examination prior to study assignment. Pre-assignment assessment of health status included an on-site veterinary examination and blood sample collection for CBC analysis, as applicable per NIRC SOPs. Animals were generally housed in pairs and allowed to acclimate for at least 3 days prior to study initiation. Groups consisted of a maximum of six animals per treatment group. All animals were immunized under ketamine HCl (10 mg / kg, IM) or Telazol (4–8 mg / kg, IM) sedation on study day 0 with a 0.5 ml dose of their respective vaccine formulation or diluent via the IM route, targeting the deltoid muscle in one forelimb of each animal. 28 days after the first immunization, a second immunization was administered to the contralateral limb of the animal.

[0212] Immunization of mice and NHPs for challenge studies Mice were inoculated with the challenge strain approximately 9–12 weeks after the last immunization. A vial of stock virus was thawed and diluted to the appropriate concentration in ice-cold sterile PBS. All mice were inoculated with 4LD 50 Equivalent to 105.54 TCID in PBS 50Mice were challenged with a total volume of 50 μl containing 1000 mg / kg Belgium09 virus. Virus challenge was performed inside a safety cabinet in an expanded ABSL2 laboratory. Mice were first anesthetized with an IP injection of ketamine / xylazine solution (ketamine 50 mg / kg and xylazine 5 mg / kg) and then challenged IN (droplets in both nostrils; 25 μl per nostril) with influenza virus in a total volume of 50 μl using a micropipette. Following the challenge procedure, mice were placed in a dorsal recumbent position and observed until recovery from anesthesia. Daily body weights were taken following H1N1 challenge. Any individual animal that showed a single >20% weight loss was euthanized. Body weight measurements were recorded daily post-challenge until euthanasia in an online database, Pristima® (Version 7.5.0 Build 8), or recorded on a study-specific worksheet.

[0213] Blood collection In mice, blood was collected from all animals under sedation by submandibular or retro-orbital venous bleed (antemortem bleed, approximately 200 μl before the study and on study days 14, 28, and 42) and cardiac puncture (terminal bleed, day 56). Mice were bled before the study to obtain baseline preimmune serum samples for prescreening purposes. For serum processing, blood samples were collected in SST tubes and allowed to clot at room temperature for 30 minutes to 1 hour. Samples were then centrifuged at 1000-1300 g for 5-10 minutes with the brake off. Serum was collected using a P200 pipettor, aliquoted into two 0.5 ml cryovials, and stored at -20°C. All bleeds were documented in a specimen collection and processing log, indicating the time of sample collection and the technician performing the procedure. Portions of serum samples were evaluated for antibody titers in HAI or ELLA and ELISA assays.

[0214] NHPs were bled for serum isolation under anesthesia using ketamine 10 mg / kg / acepromazine 1 mg / kg administered intramuscularly (days -4, 2, 7, 14, 28, 30, 35, 42, 56, 90, and 180). Blood volume did not exceed established guidelines for percentage of body weight and animal health. Blood was collected from anesthetized NHPs using femoral vein puncture using 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 isolated by spinning the tube at 1200 × g for 10 minutes at room temperature. Serum was then aliquoted into labeled cryovials (1 ml / vial) and stored at ≤ -20°C. Portions of serum samples were evaluated for antibody titers in HAI or ELLA and ELISA assays. For PBMCs, NHPs were prebled before vaccination and again approximately 42–63 days after the first injection. For this purpose, blood was collected in BD Vacutainer® tubes containing heparin anticoagulant. Briefly, anticoagulated blood samples were diluted in PBS and subjected to gradient density centrifugation at 400 × g for 30 minutes using Histopaque® separation medium (Sigma). The opaque interface containing mononuclear cells was then collected and washed three times with PBS using low-speed (250 × g) centrifugation, with a final centrifugation to reduce the platelet count. Live versus dead PBMCs were enumerated using a Nexcelom Cellometer K2. PBMCs were cryopreserved in FBS with 10% DMSO using Mr. Frosty® freezing boxes. The boxes were immediately placed in a -80°C freezer 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 on 96-well high-binding polystyrene plates at a concentration of 2 μg / ml in carbonate-bicarbonate buffer. Plates were collected and incubated overnight (16 ± 4 h) at 2–8°C. After overnight incubation, the antigen-coated plates were washed five times with wash buffer (PBS, 0.5% Tween 20) and blocked with blocking solution (10% BSA in PBS) for 60 ± 30 min at room temperature. Test samples, naive controls, and reference samples were diluted in sample diluent (PBS, 10% BSA, 0.5% Tween 20) and added in duplicate to wells, followed by incubation for 90 min at room temperature. Plates were washed five times with wash buffer, and goat anti-mouse HRP was added for mouse sera and goat anti-monkey HRP for NHP sera at a dilution of 1:10,000. The plates were then incubated at room temperature for 30 minutes, and excess HRP-IgG was washed away 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 >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 a 30-minute incubation period at 56°C, followed by the addition of 6 parts PBS to achieve 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 every assay plate. Each plate also included a back titration to verify the antigen dose (4 HAU / 25 μl) as well as a negative control sample (PBS or naive control serum). The HAI titer was determined as the highest dilution of serum resulting in complete inhibition of hemagglutination. Results were valid only for plates with appropriate back titration results (demonstrating a 4 HAU / 25 μl loading) and a reference serum titer within 2-fold of the expected titer.

[0217] NAI assay Neuraminidase inhibition (NAI) antibody titers were determined using a method for enzyme-linked lectin assay (ELLA). The antigen source (viral NA) was titrated, and a standard amount was 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 a standard amount of virus was added in duplicate to wells of fetuin-coated plates. The 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 the washed plates and incubated at room temperature for 2 hours. Color development was achieved by adding substrate (ODP in sodium citrate) and incubating for 10 minutes. The reaction was then terminated by adding stop buffer (1N sulfuric acid). Plates were scanned for absorbance at OD 490 nm. A decrease or absence of color compared to the virus control indicated inhibition of NA activity due to the presence of NA-specific antibodies. NAI titer (IC50 The IC50 value was calculated from the OD readings and the results were graphed in GraphPad Prism. If the ELLA titration curve did not provide a good enough 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 prewarmed in a 37°C water bath. PBMCs were rapidly thawed in a 37°C water bath and transferred dropwise into a conical tube with prewarmed medium. The tubes were centrifuged at 1,500 rpm for 5 minutes, and the cells were resuspended and counted using a Guava cell counter. A monkey IFN-γ ELISPOT kit (Mabtech 3421M-4APW) and an IL-13 ELISPOT kit (Mabtech 3470M-4APW) were used. Pre-coated plates provided with the kit were washed four times with sterile PBS and blocked with 200 μl of complete medium for at least 30 minutes in a 37°C incubator. The 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) at 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, the cells were removed, the plates were 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 a 2-hour incubation, the plates were washed five times with PBS and incubated with 100 μl of 1:1000 diluted streptavidin in each well for 1 hour at room temperature. The plates were developed with 100 μl of BCIP / NBT substrate solution until spots appeared. The plates were rinsed with tap water, air-dried, scanned, and enumerated using a CTL ImmunoSpot® reader (Cellular Technology Ltd.). Data were reported as spot-forming cells (SFC) per million PBMCs.

[0220] Memory B cell (MBC) ELISPOT assay Sing16 H3-specific and total IgG were measured using a human IgG Single-Color Memory B Cell ELISPOT kit (CAT#NC1911372, CTL) according to the manufacturer's instructions. + 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), transferred to a tube containing prewarmed 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 diluted to 2 × 10 per ml in 5 ml of complete medium. 6The cells were resuspended in PBS and transferred to a T25 flask for 1 hour in a 37°C, 5% CO2 incubator. The volume of the cell suspension was then adjusted to 6 ml, and B-Poly-S was added at a 1:1000 dilution. The cells were left in the CO2 incubator for 4 days of stimulation. The kit-supplied PVDF microplates were prewetted with 70% ethanol, rinsed, and coated overnight with 80 μl / well of the kit-supplied anti-human IgG capture Ab or 4 μg / ml of Sing16 / H3 recombinant protein.

[0221] Cells were harvested 4 days after stimulation, washed, counted, and adjusted to the indicated concentrations in CM. Coated microplates were washed with PBS, blocked with CM for 1 hour, and emptied. 100 μl / well of cell suspension was added to the plate and incubated at 37°C in a CO2 incubator 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, 80 μl / well of a 1:1000 diluted streptavidin-AP was added to the plate for 1 hour. Freshly prepared substrate solution was added and incubated at room temperature for 18 minutes. Plates were rinsed with tap water, air-dried, scanned, and enumerated using a CTL ImmunoSpot® reader (Cellular Technology Ltd.). For each individual animal, IgG + The number of antigen-specific ASCs and Sing16 / H3-specific ASCs was calculated per million PBMCs. + Calculated as % of antigen-specific ASC to ASC. To assess assay background, negative control wells on all plates were coated with PBS (no background detected).

[0222] statistical analysis Shining T max To estimate T for individual subjects based on observed data, nonparametric methods are used. maxwas estimated. To estimate the half-life of luminosity, a linear model was fitted to the log-transformed data for each subject during the time course of decay from maximum luminosity to baseline, assuming an exponential decay model for luminosity after reaching maximum (we estimate baseline using the mean luminosity in the saline group). The half-life was estimated as the time point at which log luminosity reached the midpoint between maximum and baseline values. For analyses of different readouts with results summarized as geometric means, SE model-based geometric means and SEs were estimated from a mixed-effects model for repeated measures where the response was the log-transformed readout, vaccination was a fixed effect, and time was the repeated measure; the log-based mean and SE estimates from the model were then transformed back to obtain geometric means and SEs. For weight change, over-descriptive statistical analysis was used. The median and range for each group of maximum % weight loss over time from baseline (day 0) was reported to assess worse-case scenarios; the median and range for each group of % weight change from baseline at the last observation was reported to assess weight recovery.

[0223] Antigen sequence The sequence of the Perth09 N2 antigen used here is MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKLAEYRNWSKPQCDITGFAPFSKDNSIRLSAGGDIWVTRE PYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSKEILRTQESECVCINGTCT VVMTDGSASGKADTKILFIEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAFDDGNDVWMGRTISEKSRLGYETFKVIEGWSNPKSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI* (SEQ ID NO: 4) is.

[0224] The sequence of the Mich15 N1 antigen used here is MNPNQKIITIGSICMTIGMANLQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIRE PFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSC FTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK* (SEQ ID NO: 5) is.

[0225] The sequence of the Sing16 H3 antigen used here is *(SEQ ID NO: 6) is.

[0226] The sequence of the Sing16 N2 antigen used here is MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVTRE PYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLIDSVVSWSKDILRTQESECVCINGTCT VVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAFDDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADLNLMHI* (SEQ ID NO: 7) is.

[0227] The sequence of the CA09 H1 antigen used here is *(SEQ ID NO: 24) is.

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

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

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

[0231] The sequence of the Perth / 16 / 2009 (H3N2) NA antigen mRNA open reading frame (ORF) used here is: is.

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

[0233] The sequence of the A / Tasmania / 503 / 2020 antigen mRNA open reading frame (ORF) used here is: is.

[0234] The sequence of the B / Washington / 02 / 2019 antigen mRNA open reading frame (ORF) used here is: is.

[0235] The sequence of the B / Phuket / 3073 / 2013 antigen mRNA open reading frame (ORF) used here is: is.

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

[0237] [Table 5]

[0238] Cryo-electron microscopy (cryo-TEM) images of CA09 HA mRNA-LNPs showed uniform spherical particles with a multilayered inner core structure. The layered nature of the solid core structure, further analyzed by Fourier transform, revealed a 3.7 nm periodicity between layers. The uniform particle morphology seen in the micrographs indicates a homogeneous LNP formulation with properly assembled LNPs.

[0239] Antigen expression was confirmed by flow cytometry by transiently transfecting human skeletal muscle cells (HskMCs) with unencapsulated mRNA constructs of CA09 HA, Sing16 HA, Sing16 NA, or Mich15 NA, followed by staining with protein-specific antibodies for analysis. High levels of HA and NA expression were observed in HskMCs, confirming the proper assembly and transport of native HA trimers and NA tetramers upon expression in muscle cells. To study the subcellular localization of the expressed HA and NA proteins, HeLa cells were transfected with bivalent H3N2 LNPs, and the proteins were visualized by immunostaining and confocal microscopy. Staining of permeabilized cells with antibodies against the corresponding proteins and the endoplasmic reticulum (ER) marker calnexin revealed that the NA signal strongly colocalized with the ER (approximately 90%), whereas HA only moderately colocalized with the ER (25%). This is consistent with the understanding that early NA and HA proteins are translocated to the ER for assembly (Dou et al., Front Immunol. (2018) 9:1581).

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

[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 have antiviral functions, and both antigens are considered important for an effective influenza vaccine (Krammer et al., Nat Rev Immunol. (2019) 19(6):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 2, 0.4, 0.08, or 0.016 μg of mRNA-LNP was administered 4 weeks apart. Total IgG responses were assessed by ELISA using recombinant HA (rHA) antigen from the same strain. HA-specific antibodies were detected in all groups after the first dose, with titers peaking 42 days after the second dose (Figure 12). To measure functional antibodies, hemagglutination inhibition (HAI) responses were assessed against the homologous strains CA09 and Sing16. Although HAI titers after the first dose were observable for the 2 μg dose CA09-LNP and Sing16-LNP treatment groups, with GMTs of 160 and 70, respectively, on day 28, a more significant increase in HAI titers was observed after the second dose. On day 42, GMT titers were 80 and 2200 for the 0.016 μg and 0.4 μg groups, respectively, for CA09-HA-LNP, and 14 and 100 for the 0.016 μg and 0.4 μg groups, respectively, for the Sing 16 HA-LNP groups (FIG. 13).

[0242] Similarly, to test anti-NA responses, mice were immunized with 2, 0.4, 0.08, or 0.016 μg of Sing16 NA-LNP or Mich15 NA-LNP. ELISA using recombinant NA antigen was performed to evaluate the total IgG response induced by the Mich15 NA-LNP or Sing16 NA-LNP formulations. Animals developed high antibody-binding responses after a single dose, and NA-binding antibodies significantly increased 42 days after the second dose (Figure 14). An enzyme-linked lectin assay (ELLA) was used as a surrogate for functional antibody titers for neuraminidase inhibitory (NAI) activity against H6N1 or H6N2 chimeric viruses. Although two doses of vaccine substantially increased functional antibody responses compared with a single dose, even at doses as low as 0.016 μg, robust NAI titers with GMTs of 800 and 60 were recorded 28 days after a single dose for Mich15 NA-LNP and Sing16 NA-LNP, respectively. On day 42, in the Sing16 NA-LNP group, GMT titers were 900 and 10,200 between 0.4 μg and 0.016 μg, respectively, indicating a dose-dependent response, with titers exceeding the ULOQ for Mich15 NA-LNP (Figure 15).

[0243] Protection from virus challenge in mice To test the efficacy of the mRNA vaccine in a murine influenza virus challenge model, we inoculated BALB / c mice IM with 0.4 μg of CA09 HA-LNP along with two doses of LNP dilution buffer as a negative control at weeks 0 and 4. HAI titers of vaccine group serum samples on study days 0, 14, 28, 42, 56, 92, and 107 demonstrated a robust immune response with GMTs of 1660 and 1:830 on days 56 and 92, respectively (Figure 16A). On day 93, all mice were challenged with a 4x dose (4x LD) of Belgium09 virus homologous to CA09, capable of causing a 50% lethal outcome. 50) intranasally. All vaccine group mice survived the challenge with no mortality and some mild morbidity characterized by transient weight loss of less than 5% (Figure 16B). However, mice in the dilution control group suffered significant and rapid weight loss, which led to high mortality (90%) by day 9. These results demonstrate the high efficacy of the HA-based MRT formulation in a lethal mouse influenza challenge model.

[0244] To evaluate the protective efficacy of the NA-based MRT vaccine, we performed a similar challenge experiment in BALB / c mice. Because the Mich15 NA-LNP vaccine elicited robust NAI titers after a single immunization in naive mice (Figure 16A), we evaluated a one- or two-dose regimen of 0.4 or 0.016 μg of Mich15 NA-LNP administered over a 4-week interval. Control groups were vaccinated with the same regimen and received 0.6 μg of hEPO-LNP or diluent buffer. Robust NAI titers were observed after a single dose, with GMTs of 14,000 NAI for 0.4 μg and 1,800 NAI for 0.016 μg of Mich15 NA-LNP recorded on day 28 (Figure 17A). After the second immunization on day 42, NAI titers increased 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 Belgium09 H1N1 virus. Individual weight changes from baseline over time by treatment group are shown graphically in Figure 17B. All mice in the two control groups suffered significant morbidity, and all animals had to be euthanized due to >20% weight loss by day 8 post-infection. Notably, all but one animal in the vaccine group survived the challenge in the single 0.016 μg group, demonstrating high protective efficacy against death even after a single dose of only 0.016 μg Mich15 NA-LNP. Although a higher dose (0.4 μg) demonstrated high overall protection, in contrast to HA immunization, NA vaccination was not sufficient to protect against weight loss, as vaccinated animals showed a moderate weight loss of 10% of their initial body weight, consistent with findings reported for other NA vaccines. Weight regain was observed for the vaccinated groups, resulting in a mean final weight change of 2.7% at the low dose and 4.8% at the higher dose compared to baseline. Overall, the results demonstrated that a single low-dose MRT NA-LNP vaccination can induce measurable functional antibodies to block influenza NA activity and sufficient to protect against lethal challenge in mice.

[0245] Immunogenicity of HA(H3)mRNA-LNPs in NHPs To evaluate the immunogenicity of mRNA-LNP in NHPs, a dose-ranging study was conducted in NHPs using 15, 45, 135, and 250 μg of Sing16 HA-LNP. After the first immunization, all vaccinated NHPs developed antibodies reactive to the recombinant HA protein as measured by ELISA (Figure 18). A further boost in titers was observed after the second dose. Surprisingly, the 15 μg dose induced ELISA titers that were only 1.8-fold lower than those at the 135 μg dose level (95% CI 1.0, 3.6), suggesting dose saturation was near the 15 μg level. Robust HAI antibodies were induced in all dose groups on day 42, with recorded GMTs of 400 for 15 μg, 700 for 45 μg, 900 for 135 μg, and 570 for 250 μg. On day 42, the fold increase in GMT titer with 95% CI was 2.2-fold (1.0; 5.0) for the 135 μg to 15 μg group and 1.3-fold (0.6; 2.8) for the 135 μg to 45 μg treatment group, indicating minimal differences between groups despite an observed trend toward higher titers with increasing dose (Figure 19A). Neutralization potency, as assessed by microneutralization (MN) assay (Figure 19B), showed a trend toward a dose effect, with GMTs of 40 for 15 μg, 180 for 45 μg, and 300 for 135 μg on day 28.

[0246] Because 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):1305–1312; Sridhar et al., Front Immunol. (2016) 7:195), we evaluated recall T cell activity in NHPs vaccinated with 45, 135, or 250 μg of Sing16 HA-LNP or 45 μg of recombinant HA. 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 sequence of Sing16 HA. All vaccinated animals except one in the 250 μg group expressed IFN-γ-secreting cells, ranging from 28 to 1328 spot-forming cells (SFC) per million PBMCs (Figure 20A). Notably, no dose response was observed, with animals at lower and higher dose levels exhibiting comparable frequencies of IFN-γ-secreting cells. In contrast, all control animals immunized with recombinant Sing16 HA protein demonstrated an 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 strong Th1-biased cellular response in NHPs, comparable to the cellular response seen with the SARS-CoV-2 vaccine currently under development, MRT5500 (Kalnin et al., supra).

[0247] To investigate the frequency of memory B cells (MBCs) in NHPs after immunization with Sing16 HA-LNPs, an ELISPOT assay was developed to quantify antigen-specific MBCs as a readout of humoral immune memory. On day 180, PBMCs were collected from NHPs immunized with either 45 μg or 15 μg formulations of Sing16 HA mRNA-LNPs or with recombinant HA as a comparator at the 45 μg dose. A 4-day polyclonal stimulation of PBMCs, optimized to drive memory B cells to antibody-secreting cells (ASCs), was performed, and the stimulated PBMCs were plated in antigen-specific ELISPOT assays, where the frequency of antigen-specific ASCs could be determined. Antigen-specific memory B cells were then 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% to 5% in the 45 μg dose group and 0.3% to 1.5% in the 15 μg dose group. In rHA-immunized animals, memory B cell responses appeared significantly lower because antigen-specific memory B cells were undetectable in five of six animals (Figure 21). It was concluded that Sing16 HA-LNP, like other mRNA vaccines, elicits a population of anti-HA-specific memory B cells that may extend population immunity (Lindgren et al., Front Immunol. (2019) 10:614).

[0248] Polyvalent influenza virus antigens An 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 examine influenza antigen combinations, co-encapsulated HA and NA mRNAs were formulated into LNPs in bivalent formulations containing 0.2 μg of each mRNA for H3H1, H3N2, or N1N2 combinations, or in monovalent formulations containing 0.2 μg of each corresponding antigen. These formulations were administered to mice to determine any antigen interference on immunogenicity by comparing the functional titers of the individual antigens in bivalent versus monovalent formulations (Figure 22, panels (a)-(c) and Table 6).

[0249] [Table 6]

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

[0251] Tetravalent formulations of co-encapsulated H1, N1, H3, and / or N2 mRNA were further investigated. These formulations were tested in NHPs at 10 μg total loads consisting of 2.5 μg of each influenza antigen mRNA and, where required in combination, non-coding mRNA (nc mRNA), resulting in tetravalent (H1N1H3N2), bivalent (H1N1 or H3N2), or monovalent (H1, H3, N1, or N2) LNPs (Table 7).

[0252] [Table 7]

[0253] HAI titers against H1 or H3, or NAI titers against N1 or N2, were compared between monovalent versus bivalent or tetravalent formulations (Figure 23). On day 42, HAI titers against H1 in the tetravalent group were comparable when analyzed with those in the H1 monovalent group (p=0.9054, t-test, unpaired, two-tailed) or H1N1 bivalent group (p=0.8002). Similarly, H3 HAI titers in the tetravalent group were comparable when analyzed with those in the H3 monovalent group (p=0.2504) or H3N2 bivalent group (p=0.5894). NAI titers against 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 titers between N2 monovalent mRNA (p=0.8485) or H3N2 bivalent mRNA (0.4545) and tetravalent H1N1H3N2 mRNA formulations.

[0254] Overall, these findings indicate that the HA / NA mRNA-LNP co-encapsulated or combination multivalent vaccine at this dose level can deliver all four antigens without concerns about antigenic interference, and all antigens were as immunogenic as in formulations where these antigens were delivered alone. [Example]

[0255] Additional LNP formulations Additional LNP formulations for mRNA vaccines were prepared and designated 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 the test mRNA. hEPO expression was measured by ELISA from samples collected from mice injected with LNPs. Samples were collected 6, 24, 48, and 72 hours after injection. As shown in Figure 24, hEPO expression was consistently higher at all time points for the 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 control LNPs containing MC3 cationic lipid.

[0257] [Table 8]

[0258] The same hEPO mRNA-LNP formulations were then tested in non-human primates (NHPs). Samples were taken 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-encoding mRNA-LNP formulations were also tested in NHPs. NHPs were administered the LNP formulations at 10 μg by intramuscular injection, and samples were collected 28 and 42 days after injection. HAI titers were measured as described above. As shown in Figure 26, each LNP formulation produced HAI titers comparable to or higher than the MC3 control formulation.

[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 of the LNP formulations produced HAI titers comparable to or higher than the MC3 control formulation.

[0261] As shown in Figure 28, HAI titers using the Sing16 H3 antigen were elevated with LNP formulations Lipid C and Lipid D. [Example]

[0262] Respiratory syncytial virus (RSV) F protein-encoding mRNA LNP formulation The effect of different cationic lipids in LNPs was tested on LNP-encapsulated RSV F protein mRNA. LNP formulations of lipid A, lipid B, lipid C, lipid D, and lipid E ​​were tested. Each LNP was composed of 40% of one of five cationic lipids, 30% of the phospholipid DOPE, 1.5% of the PEGylated lipid DMG-PEG2000, and 28.5% cholesterol. LNPs containing the cationic lipid MC3 were also used and are considered an industry benchmark (Jayaraman et al., Angew Chem Int Ed. 51:8529-33. 2012).

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

[0264] FD3 (SEQ ID NO: 16)

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

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

[0267] FD3 mRNA ORF:

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

[0269] FD3 DNA:

[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:

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

[0276] As shown in Figure 30, all cationic lipids tested produced effective RSV neutralization titers to levels similar to Pre-F NP with aluminum adjuvant.

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

[0278] [Table 9]

[0279] [Table 10]

[0280] A better immune response is indicated by a lower antibody / neutralization titer ratio. Here, the LNP formulation Lipid B showed the best immune response, and all LNP formulations showed superior immune responses compared to the non-mRNA vaccine, Pre-F NP, and some even better than the industry benchmark LNP formulation MC3. [Example]

[0281] SARS-CoV-2 spike (S) protein-encoding mRNA LNP formulation LNP formulation containing SARS-CoV-2 spike (S) protein-encoding mRNA Human subjects were administered an LNP formulation containing mRNA encoding the SARS-CoV-2 S protein. Subjects received LNPs in formulation lipid B. The unmodified mRNA encoded a SARS-CoV-2 S protein mutated to remove the furin cleavage site and mutate residues 986 and 987 to proline. Subjects received the LNP-SARS-CoV-2 vaccine under the clinical trial protocol for NCT04798027 described below.

[0282] This was a sequential cohort prevention study consisting of a sentinel cohort followed by a full enrollment cohort. The sentinel cohort had three dose levels (up to 25 participants aged 18-49 years for each dose level), which were conducted in an open-label fashion with tiered safety assessments for each dose level and each vaccination. All sentinel participants received two doses of the vaccine, 21 days apart. The full enrollment cohort was stratified into two age groups based on age at enrollment: a young adult age group (140 participants aged 18-49 years) and an elderly age group (168 participants aged ≥50 years). Full enrollment cohort 1 (groups 1-4) received a single injection of the study intervention, while cohort 2 (groups 5-8) participants received two doses of the vaccine (given 21 days apart). The route of administration for all groups 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] Study results showed neutralizing antibody seroconversion (defined as a four-fold increase over baseline) in 91% to 100% of study participants across all three doses tested, two weeks after the second injection. No safety concerns were observed, and the tolerability profile is comparable to other unmodified mRNA SARS-CoV-2 vaccines. [Example]

[0292] Further studies on quadrivalent or octavalent influenza vaccine LNP formulations HAI and NAI titers were measured in mice administered various multivalent 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 for mice receiving monovalent or tetravalent HA or NA mRNA vaccines.

[0294] Mice were injected with a prime vaccine on day 0 and an equal dose of a booster vaccine on day 21. Blood was collected on days 1, 20, 22, and 35. Monovalent compositions containing mRNA encoding HA or NA antigens used mRNA encoding each of the following individually: H1, H3, 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). A tetravalent vaccine composition was also prepared containing mRNA encoding N1, N2, and NA from the B / Victoria lineage and NA from the B / Yamagata lineage, as well as 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 / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013). Finally, an octavalent vaccine composition 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) was prepared and administered as an octavalent vaccine. Each mRNA was added at 0.4 μg per strain for all compositions. Each group contained six mice.

[0295] A summary of each experimental group is listed in Table 11 below.

[0296] [Table 11]

[0297] As shown in Figure 31, the octavalent mRNA-LNP formulation produced HAI titers within four-fold of the tetravalent 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 formulation produced NAI titers comparable to the tetravalent mRNA-LNP formulation.

[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 HAs from four different influenza strains does not appear to suppress or interfere with anti-NA immune responses.

[0300] High-content imaging-based neutralization test (HINT) titers for HA and NAI titers were also measured from ferrets administered various multivalent 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 polyvalent vaccine immunogenicity were vaccinated twice, 21 days apart, with either (1) a mixture of four mRNAs encoding NA antigens (N1, N2, BvNA, and ByNA), (2) a mixture of four mRNAs encoding HA antigens (H1, H3, BvHA, and ByHA), or (3) a mixture of four mRNAs encoding NA antigens (N1, N2, BvNA, and ByNA) and four mRNAs encoding HA antigens (H1, H3, BvHA, and ByHA), as shown in Table 12 below. Each HA contained HA from one of 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 at a 1:1 ratio.

[0302] A summary of each experimental group is listed in Table 12 below.

[0303] All ferrets were bled under sedation (isoflurane) at baseline, 1 day or immediately prior to the booster, at the time of booster vaccination, and 2 weeks post-challenge, as required. Serum samples (stored at -20°C until required) were tested by ELLA to assess NAI activity. Additionally, a hemagglutination inhibition assay (HAI) was undertaken to assess antibody responses to hemagglutinin antigens following polyvalent 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 formulation produced HINT titers comparable to the tetravalent mRNA-LNP formulation.

[0306] A summary of the NAI titer results for each of the above groups is shown in Figure 34 (day 20) and Figure 35 (day 42). The octavalent mRNA-LNP formulation produced NAI titers comparable to the tetravalent mRNA-LNP formulation. This was true for day 20 through day 42 samples. [Example]

[0307] Functional antibody titers against influenza heterosubtype strains recorded using mRNA in lipid A or lipid B LNP formulations To evaluate the immunogenicity of mRNA-LNPs in NHPs, they were immunized with 0, 15, or 45 μg of Sing16 HA-encoding mRNA (encoding HA A / Singapore / INFIMH-16-0019 / 2016) encapsulated in lipid A or lipid B LNP formulations. Naive male and female cynomolgus macaques (Macaca fascicularis) originating from Mauritius were used. At the start of the study, animals weighed >2 kg and were >2 years old. Groups consisted of up to six animals per treatment group. On study day 0, each animal was vaccinated with 0.5 mL of the respective vaccine dose or dilution via the intramuscular route in one forelimb, targeting the deltoid muscle. 28 days after the first immunization, the animals received a second immunization in the contralateral limb. A tetravalent egg-derived inactivated influenza vaccine (IIV) containing the A / Singapore / INFIMH-16-0019 / 2016 (H3N2) strain was used as the comparator.

[0308] Influenza assays were performed using the A / Singapore / INFIMH-16-0019 / 2016 (H3N2) virus stock from BIOQUAL, Inc. Additional breadth 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 from both the 3c.2a and 3c.3a clades, as well as a very distantly related swine-like H3 sequence (A / Michigan / 84 / 2016) based on bioinformatics analysis to select the maximally diverse set of H3N2 sequences from the same time frame as A / Singapore / INFIMH-16-0019 / 2016.

[0309] For the microneutralization (MN) assay, serum samples were diluted in receptor-destroying enzyme (Denka Seiken, 370013) and incubated overnight in a 37°C water bath. Samples were heat-inactivated at 56°C for 30 minutes, and then two-fold serial dilutions were run in duplicate on a 96-well plate. 100 TCID 50 An equal volume of virus was added to the plate at 100 μl, followed by incubation at 37°C for 1 hour. One hundred 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 with 5% CO2 for 48 hours. The plate was fixed with cold acetone and then stained with biotin-conjugated anti-influenza A NP (Millipore, MAB8258B), followed by incubation 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 developed neutralizing antibodies against the homologous virus, A / Singapore / INFIMH-16-0019 / 2016 (H3N2), as documented by MN assay (Figures 36 and 37). Furthermore, in this model, contrary to IIV vaccines, MN titers were observed against a heterosubtypic virus panel including A / Shandoglaicheng / 1763 / 2016, A / Louisiana / 13 / 2017, A / Kenya / 105 / 2017, A / Victoria / 746 / 2017, and A / Aksaray / 4048 / 2016. The data demonstrate the potential of the mRNA formulation to offer greater breadth than IIV in substituting for heterosubtypic strains of influenza.

Claims

1. 1. A pharmaceutical composition comprising a nucleic acid molecule encapsulated in a lipid nanoparticle (LNP), wherein the LNP comprises: cationic lipid GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 45%, a polyethylene glycol (PEG) conjugated (PEGylated) lipid, the PEGylated lipid being DMG-PEG2000, at a molar ratio of 0.25% to 2.75%; a cholesterol-based lipid, the cholesterol-based lipid being cholesterol, in a molar ratio of 20% to 35%; and a helper lipid, the helper lipid being 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), in a molar ratio of 25% to 35%; Here, GL-HEPES-E3-E12-DS-4-E10 is 【Chemistry 1】 And, And all of the molar ratios are relative to the total lipid content of the LNP.

2. LNP is GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30% The composition of claim 1 comprising:

3. The composition of claim 1, wherein the LNPs have an average diameter of 30 to 200 nm or 80 to 150 nm.

4. 2. The composition of claim 1, wherein the nucleic acid molecule(s) is an mRNA molecule comprising an open reading frame (ORF).

5. The composition of claim 4 , wherein the mRNA molecule encodes an antigen.

6. The composition of claim 4 , wherein the antigen is derived from an influenza virus.

7. The composition of claim 4 , wherein the LNP comprises two or more mRNA molecules, each mRNA molecule encoding a different antigen.

8. The composition of claim 7, comprising two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding (i) one or more hemagglutinin (HA) antigens, (ii) one or more neuraminidase (NA) antigens, or (iii) at least one HA antigen and at least one NA antigen.

9. 7. The composition of claim 6, comprising one or more mRNA molecules encoding antigens of influenza A, B and / or C viruses.

10. 5. The composition of claim 4, wherein the mRNA molecule comprises an open reading frame (ORF) encoding a respiratory syncytial virus (RSV) F protein antigen.

11. The composition of claim 4, wherein the ORF is codon-optimized.

12. 10. The composition of claim 1 formulated for intramuscular injection.

13. 9. The composition of claim 8, wherein the antigen comprises an influenza virus HA antigen and / or an influenza virus NA antigen having a molecular sequence identified or designed from a machine learning model.

14. The composition of claim 1, wherein the composition comprises 1 to 10 mg / mL of LNP.

15. The composition of claim 1, wherein the LNP comprises 1 to 20 nucleic acid molecules.

16. The composition of claim 5 , wherein the antigen comprises a viral antigen or a bacterial antigen.

17. The composition of claim 7, wherein the different antigens are from the same pathogen.

18. 8. The composition of claim 7, wherein the different antigens are from different pathogens.

19. The composition of claim 4 , wherein the composition comprises two or more LNPs, each LNP comprising mRNA encoding a different antigen.

20. 20. The composition of claim 19, wherein the different antigens are from the same pathogen.

21. 20. The composition of claim 19, wherein the different antigens are from different pathogens.

22. 10. The composition of claim 9, wherein the antigen is an HA and / or NA antigen of influenza A and influenza B viruses.

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

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

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

26. The composition of claim 22, comprising 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 strain, and one mRNA molecule encoding an HA antigen from influenza B / Victoria.

27. The composition of claim 22, 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 from the influenza B / Yamagata lineage, one mRNA molecule encoding an NA antigen from the influenza B / Yamagata lineage, one mRNA molecule encoding an HA antigen from the influenza B / Victoria lineage, and one mRNA molecule encoding an NA antigen from the influenza B / Victoria lineage.

28. 11. The composition of claim 10, wherein the RSV F protein antigen comprises or consists of an amino acid sequence having at least 98% identity to SEQ ID NO:

16.

29. 11. The composition of claim 10, wherein the RSV F protein antigen is in a prefusion conformation.

30. The composition of claim 10, wherein the mRNA comprises a nucleic acid sequence having at least 80% identity to the nucleic acid sequence set forth in SEQ ID NO:

17.

31. The composition of claim 10, wherein the mRNA comprises a nucleic acid sequence having at least 80% identity to the nucleic acid sequence set forth in SEQ ID NO:

21.

32. The mRNA contains the following structural elements: (i) a 5′ cap having the following structure: 【Chemistry 2】 (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 19; (iii) a protein coding region having the nucleic acid sequence of SEQ ID NO: 17; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 20; and (v) poly(A) tail The composition of claim 10 comprising:

33. The composition of claim 4, 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 polyadenylation (poly(A)) sequence.

34. The composition of claim 4 , wherein the mRNA comprises at least one chemical modification.

35. 5. The composition of claim 4, 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.

36. The composition of claim 4, 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.

37. 35. The composition of claim 34, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-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.

38. 13. The composition of claim 12, comprising phosphate buffered saline and / or comprising trehalose.

39. 13. The composition of claim 12, comprising phosphate buffered saline and / or trehalose at 10% (w / v) of the composition.

40. A kit comprising a container containing a single-use or multi-use dosage of the composition of claim 1.

41. 41. The kit of claim 40, wherein the container is a vial or a prefilled syringe or injector.

42. providing an aqueous buffered solution containing nucleic acid molecules; providing an amphiphilic solution comprising a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid; and Mixing the aqueous buffer solution with the amphiphilic solution in a ratio of 5:1 to 3:1, and in some cases 4:

1.

10. A method for making the composition of claim 1, comprising:

43. 43. The method of claim 42, wherein the aqueous buffer solution is an acidic buffer solution.

44. 44. The method of claim 43, wherein the aqueous buffer solution comprises 1 mM citric acid and 150 mM sodium chloride at a pH of about 4.

5.

45. 43. The method of claim 42, wherein the amphiphilic solution is an ethanol solution.

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