SARS-COV-2 RNA VACCINE COMPOSITIONS AND METHODS OF USE

Abstract

The present disclosure provides compositions, methods of treatment, and methods of making and using compositions for delivering nucleic acid to a subject. The compositions described herein include a lipid carrier, optionally including inorganic particles, that can be mixed with the nucleic acid. Methods of using the compositions as COVID-19 vaccines for treating coronavirus infection are also provided. COVID-19 is an infectious respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has spread worldwide.

Description

[Technical field]

[0001] cross reference This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 247,169, filed September 22, 2021, and U.S. Provisional Patent Application No. 63 / 297,397, filed January 7, 2022, the contents of each of which are incorporated by reference in their entirety herein. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with Government support under Contract Nos. 75N93020C00052 and 75N93019C00037 awarded by the National Institute of Allergy and Infectious Diseases, Division of Microbiology and Infectious Diseases. The Government has certain rights in this invention. Sequence Listing

[0003] This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on January 21, 2021, is named 201953-713601_SL.txt, and is 372,976 bytes in size. [Background technology]

[0004] background COVID-19 is an infectious respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has spread worldwide. Although commercially available mRNA vaccines provide some protection to individuals infected with SARS-CoV-2 from COVID-19 disease, the virus is still highly contagious and can cause a wide range of symptoms even in vaccinated individuals. Furthermore, SARS-CoV-2 is known to mutate, and the efficacy of vaccines against current and novel variants of SARS-CoV-2 is still being determined. Thus, there is a great need for highly effective vaccine compositions against SARS-CoV-2 infection. Summary of the Invention [Means for solving the problem]

[0005] A quick overview Provided herein are compositions comprising a lipid carrier, the lipid carrier comprising a liquid oil; and a surfactant, the surfactant comprising a cationic lipid; a hydrophilic surfactant; and a hydrophobic surfactant; and at least one nucleic acid, the at least one nucleic acid comprising a sequence at least 85% identical to SEQ ID NOs:1-7, optionally SEQ ID NOs:1-8.

[0006] Further provided herein is a composition comprising a lipid carrier, the lipid carrier comprising a liquid oil; and a surfactant, the surfactant comprising a cationic lipid; a hydrophilic surfactant; and a hydrophobic surfactant; and at least one nucleic acid, the nucleic acid comprising a SARS-CoV-2 omicron variant spike protein antigenic sequence or a functional variant thereof.

[0007] Further provided herein is a composition comprising a lipid carrier, the lipid carrier comprising a liquid oil; and a surfactant, the surfactant comprising a cationic lipid; a hydrophilic surfactant; and a hydrophobic surfactant; and at least one nucleic acid, the nucleic acid comprising a sequence encoding an S2 region, optionally a stem helix region, of a SARS-CoV-2 spike protein antigenic sequence or a functional variant thereof.

[0008] Further provided herein is a composition comprising a lipid carrier, the lipid carrier comprising a liquid oil; inorganic nanoparticles, the inorganic nanoparticles comprising iron oxide present in an amount of about 0.2 mg / ml of 12 nm iron oxide; and a surfactant, the surfactant comprising a cationic lipid; and at least one nucleic acid, the nucleic acid comprising at least one sequence encoding a SARS-CoV-2 spike protein antigenic sequence or a functional variant thereof.

[0009] Further provided herein is a composition comprising a lipid carrier, the lipid carrier being a nanoemulsion comprising about 30 mg / mL DOTAP chloride; about 37.5 mg / mL squalene; about 37 mg / ml sorbitan monostearate; about 37 mg / ml polysorbate 80; about 10 mM sodium citrate; and about 0.2 mg Fe / ml 12 nm oleic acid coated iron oxide nanoparticles; and at least one nucleic acid, the at least one nucleic acid comprising a sequence encoding a SARS-CoV-2 spike protein antigenic sequence or a functional variant thereof.

[0010] Further provided herein is a composition comprising a lipid carrier, the lipid carrier being a nanoemulsion comprising: DOTAP chloride present in an amount of about 0.75 mg; squalene present in an amount of about 0.94 mg; sorbitan monostearate present in an amount of about 0.93 mg; polysorbate 80 present in an amount of about 0.93 mg; citric acid monohydrate present in an amount of about 1.05 mg; oleic acid coated iron oxide nanoparticles present in an amount of about 0.005 mg; and at least one nucleic acid, wherein the at least one nucleic acid comprises a sequence encoding a SARS-CoV-2 spike protein antigenic sequence or a functional variant thereof.

[0011] Further provided herein is a composition comprising a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence encoding an antigen, the antigen being a SARS-CoV-2 spike protein. Further provided herein is a vaccine comprising a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence encoding an antigen, the antigen being a SARS-CoV-2 spike protein.

[0012] Further provided herein is a method of generating an immune response in a subject, the method comprising administering to the subject a composition provided herein, thereby generating an immune response to the antigen.

[0013] Further provided herein is a method of reducing the severity of a SARS-CoV-2 infection, the method comprising administering a composition provided herein prior to infection.

[0014] Further provided herein is a method of enhancing an immune response in a subject, the method comprising administering to the subject a composition provided herein, thereby enhancing the immune response to an antigen.

[0015] Further provided herein is a method of treating a coronavirus infection, the method comprising administering a composition provided herein to a subject, thereby treating the coronavirus infection.

[0016] Further provided herein is a method of immunoprotecting a subject, the method comprising administering to the subject a composition or vaccine provided herein.

[0017] Further provided herein is a composition for immunoprotecting a subject, the composition comprising: a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and at least one nucleic acid sequence, wherein the nucleic acid sequence comprises a sequence encoding an antigen, and the antigen is a SARS-CoV-2 spike protein.

[0018] Provided herein is a dry composition comprising: a lipid carrier, where the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; at least one nucleic acid sequence, where the nucleic acid sequence comprises a sequence encoding an antigen, and the antigen is a SARS-CoV-2 spike protein; and at least one cryoprotectant.

[0019] Further provided herein is a method for preparing a lyophilized composition, the method comprising the steps of obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and lyophilizing the formulation to form a lyophilized composition.

[0020] Further provided herein is a method for preparing a spray-dried composition, the method comprising the steps of obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and spray drying the formulation to form the spray-dried composition.

[0021] Further provided herein is a method for reconstituting a lyophilized composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; (c) lyophilizing the formulation to form a lyophilized composition; and reconstituting the lyophilized composition in a suitable diluent.

[0022] Further provided herein is a method for reconstituting a spray-dried composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; (d) spray drying the formulation to form the spray-dried composition; and (e) reconstituting the spray-dried composition in a suitable diluent.

[0023] Further provided herein is a composition for the prevention of SARS-CoV-2, comprising: (a) a sorbitan fatty acid ester; (b) an ethoxylated sorbitan ester; (c) a cationic lipid; (d) an immunostimulant, and (e) at least one RNA molecule.

[0024] Further provided herein is a composition for the prevention of SARS-CoV-2, comprising: (a) sorbitan monostearate (e.g., SPAN® 60); (b) polysorbate 80 (e.g., TWEEN® 80); (c) DOTAP; (d) an immunostimulant, and (e) at least one RNA molecule.

[0025] Further provided herein is a dry composition comprising: a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core and one or more lipids; at least one nucleic acid comprising a sequence encoding a sequence capable of expressing an antigen, optionally wherein the antigen is a SARS-CoV-2 spike protein or a functional variant thereof; and at least one sugar present in an amount of (i) at least about 50% by weight of the dry composition, or (ii) at least 50 mg.

[0026] Further provided is a composition comprising a first nucleic acid comprising a sequence encoding an RNA-dependent RNA polymerase and a second nucleic acid comprising a sequence encoding a SARS-CoV-2 antigen sequence.

[0027] Further provided is a composition comprising a first nucleic acid comprising a sequence encoding an RNA-dependent RNA polymerase, and a second nucleic acid sequence encoding a SARS-CoV-2 antigen spike sequence.

[0028] Further provided is a composition comprising a first nucleic acid comprising a sequence encoding an RNA-dependent RNA polymerase and a second nucleic acid comprising a sequence encoding a SARS-CoV-2 omicron antigen sequence.

[0029] Further provided herein is a composition comprising a first nucleic acid comprising a sequence encoding an RNA-dependent RNA polymerase, and a second nucleic acid comprising a sequence encoding a SARS-CoV-2 spike S2 ​​sequence region, and optionally a stem helix region.

[0030] Further provided herein are kits that contain the compositions provided herein.

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the invention are utilized. [Brief description of the drawings]

[0032] [Figure 1]FIG. 1 shows the repRNA constructs disclosed herein. A schematic diagram of the SARS-CoV-2 spike is shown, with the signal peptide (sp), S1 and S2 domains, transmembrane domain (TM), and cytoplasmic domain (CD) indicated. The repRNAs are identified as A.1 spike, SEQ ID NO:1 encoding B.1 spike, A.1. prefusion stabilized spike, SEQ ID NO:2 encoding B.1 prefusion stabilized spike, SEQ ID NO:5 encoding B.1.1.7 prefusion stabilized spike, and SEQ ID NO:4 encoding B.1.351 prefusion stabilized spike. Mutations are shown relative to the A.1 strain (SEQ ID NO:1).

[0033] [Diagram 2] Figure 2 shows the effect of spike modifications on binding antibody responses in mice. C57BL / 6 mice (n=5 / group) were immunized with repRNA formulated in a lipid carrier encoding wild-type (WT), prefusion stabilizing (PreF), furin cleavage site deletion (Furmut), or a combination of PreF and Furmut modifications of the full-length spike of A.1 lineage SARS-CoV-2. Vaccinations were administered on days 0 and 28, and sera collected on days 14, 28, and 38 were assayed by enzyme-linked immunosorbent assay for anti-spike binding IgG.

[0034] [Figure 3-1] Figures 3A-3C show schematic diagrams of exemplary nanoparticle (NP) carriers. Figure 3A shows an oil-in-water emulsion. Figure 3B shows a nanostructured lipid carrier (NLC). Figure 3C shows a nanoparticle with inorganic nanoparticles in liquid oil. [Figure 3-2] Same as above.

[0035] [Figure 4A] Figures 4A-4B show the increase in protein production induced by NP-3 in a Miglyol lipid carrier formulation. Figure 4A shows the first assay. Figure 4B shows the second assay. [Figure 4B] Same as above.

[0036] [Figure 5A] Figures 5A-5B show the reduction in immune response induced by NP-3 in a Miglyol lipid carrier formulation. Figure 5A shows the first assay. Figure 5B shows the second assay. [Figure 5B] Same as above.

[0037] [Figure 6] Figures 6A-6B show the correlation between enhanced protein production observed with NP-3 and low TNF (e.g., TNF alpha) stimulation as a result of the first and second assays. Figure 6A shows the first assay. Figure 6B shows the second assay.

[0038] [Figure 7-1] 7A-7F show SEAP levels in BALB / c mice intramuscularly injected with various embodiments of lipid carrier formulations described herein. FIG. 7A shows SEAP levels 4 days after injection. FIG. 7B shows SEAP levels 6 days after injection. FIG. 7C shows SEAP levels 8 days after injection. FIG. 7D shows SEAP levels 4 days after injection. FIG. 7E shows SEAP levels 6 days after injection. FIG. 7F shows SEAP levels 8 days after injection. X-axis: condition, Y-axis: relative light units (RLU). [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0039] [Figure 8] FIG. 8 is a bar graph showing Z-average measurement values ​​and polydispersity index (PDI) on the Y-axis and group number on the X-axis for conditions 1 to 14.

[0040] [Figure 9]9A-9B show dot charts depicting anti-D614G IgG levels for conditions 1-14. FIG. 9A shows a dot chart with IgG (μg / ml) on the Y-axis and group number on the X-axis for conditions 1-14. Measurements for anti-D614G (1:40 dilution) IgG response were recorded on day 14. FIG. 9B shows a dot chart with IgG (μg / ml) on the Y-axis and group number on the X-axis for conditions 1-14. Measurements for anti-D614G (1:200 dilution) IgG response were recorded on day 28.

[0041] [Figure 10] FIG. 10 shows a dot chart with measured anti-D614G (1:200 dilution) IgG (μg / ml) on the Y-axis and an indication of storage conditions on the X-axis at day 28. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description of the Invention Various aspects are now described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0043] Provided herein are compositions, kits, methods, and uses thereof for inducing an immune response against coronavirus. Briefly, (1) nanoparticle carrier systems; (2) nucleic acids encoding coronavirus antigens and nucleic acids encoding RNA polymerase; (3) combination compositions; (4) thermostable, dried, and lyophilized SARS-CoV-2 RNA vaccines; (5) pharmaceutical compositions; (6) dosing; (7) administration; (8) therapeutic applications, and (9) kits are further described herein. definition

[0044] All definitions, as defined and used herein, should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meaning of the defined terms.

[0045] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, and in some cases may include the entirety of the document.All references disclosed herein, including patent and non-patent literature, are hereby incorporated by reference in their entirety, as if each were individually incorporated.However, when patents, patent applications or publications containing express definitions are incorporated by reference, it should be understood that these express definitions apply to the incorporated patents, patent applications or publications in which they are found, and do not necessarily apply to the text of this application, particularly the claims of this application, and in that case, the definitions provided herein shall mean priority.

[0046] The indefinite articles "a" and "an," as used herein in the specification and the claims, unless clearly dictated to the contrary, should be understood to mean "at least one."

[0047] The term "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjointly present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to the specifically identified elements, may optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open-ended term such as "comprising", may refer in one embodiment to only A (including elements other than B, if desired), in another embodiment to only B (including elements other than A, if desired), in yet another embodiment to both A and B (including other elements, if desired), etc.

[0048] When used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., including at least one of a number or list of elements, but also including two or more, and including additional unlisted items, if necessary. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or," when used herein, shall be interpreted merely as indicating exclusive alternatives (i.e., "either one, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0049] As used herein, "as needed" or "as needed" means that the situation described thereafter may or may not occur, such that the description includes instances where the situation occurs and instances where the situation does not occur.

[0050] As used herein, the terms "about" or "approximately" refer to a range of up to ±20% of a given value. Alternatively, particularly with respect to biological systems or processes, these terms can refer to within an order of magnitude, preferably within two-fold, of a value. When a particular value is described in the present application and claims, unless otherwise specified, the term "about" is implicit and in this context means within an acceptable range of error for the particular value.

[0051] The term "effective amount" or "therapeutically effective amount" refers to an amount that is sufficient to realize or at least partially realize a desired effect. Nanoparticle Carrier System

[0052] Provided herein are various compositions that include a nanoparticle or nanoparticles.Nanoparticles are also referred to herein as carriers or abbreviated as NP.Provided herein are nanoparticles that can be organic materials, inorganic materials, or combinations of inorganic and organic materials with diameters of less than about 1 micrometer (μm).In some embodiments, provided herein are nanoparticles that are used as delivery systems for provided herein bioactive agents.

[0053] A variety of nanoparticles and nanoparticle formulations (i.e., nanoemulsions) are used. Exemplary nanoparticles are illustrated in Figures 3A-3C. Nanoparticles provided herein may include, but are not limited to, oil-in-water emulsions, nanostructured lipid carriers (NLCs), cationic nanoemulsions (CNEs), vesicular phospholipid gels (VPGs), polymeric nanoparticles, cationic lipid nanoparticles, liposomes, gold nanoparticles, solid lipid protein nanoparticles, polyethyleneimine nanoparticles (LNPs or SLNs), mixed-phase core NLCs, ionized lipid carriers, magnetic carriers, polyethylene glycol (PEG)-functionalized carriers, cholesterol-functionalized carriers, polylactic acid (PLA)-functionalized carriers, and polylactic-co-glycolic acid (PLGA)-functionalized lipid carriers.

[0054] An oil-in-water emulsion, as shown in FIG. 3A (not to scale), is a stable immiscible fluid that contains oil droplets dispersed in water or in an aqueous phase. FIG. 3B (not to scale) illustrates a nanostructured lipid carrier (NLC) that may include a mixture of solid organic lipids (e.g., trimyristin) and liquid oil (e.g., squalene). In an NLC, the solid lipid is dispersed in the liquid oil. The nanodroplets are dispersed entirely in the aqueous phase. In some embodiments, the nanoparticles include inorganic nanoparticles as solid inorganic nanoparticles (e.g., iron oxide nanoparticles) dispersed in the liquid oil, as shown in FIG. 3C (not to scale). Thus, the nanodroplets are dispersed entirely in the aqueous phase as colloids. In some embodiments, the nanoparticles provided herein are dispersed in an aqueous solution. Non-limiting examples of aqueous solutions include water (e.g., sterile water, distilled water, deionized water, ultrapure water, RNase-free water, etc.), saline solutions (e.g., Kreb's saline, Ascaris saline, Dent's saline, Tet's saline), or 1% (w / v) dimethyl sulfoxide (DMSO) in water.

[0055] In some embodiments, the nanoparticles provided herein comprise a hydrophilic surface. In some embodiments, the hydrophilic surface comprises a cationic lipid. In some embodiments, the hydrophilic surface comprises an ionized lipid. In some embodiments, the nanoparticles comprise a membrane. In some embodiments, the membrane comprises a cationic lipid. In some embodiments, the nanoparticles provided herein comprise a cationic lipid. Exemplary cationic lipids for inclusion into hydrophilic surfaces include, but are not limited to, 1,2-dioleoyloxy-3(trimethylammonium)propane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]N,N,N trimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-di methylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,1'-((2-(4-(2-((2-(bis(2-hydroxy-dodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 306Oi10, tetrakis(8-methylnonyl)3 ,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate; 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate;ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17 -tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol; BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-cholesterol, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10, 11,12,13,14,15,16,17-Tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide; cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine;FTT5, hexa(octan-3-yl) 9,9',9'',9''',9'''',9'''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate; Lipid H (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane- 4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate); PEG2000-DMG, (R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)carbamate; TT3, or N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide. Other examples of suitable classes of lipids include, but are not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG); and PEGylated lipids (e.g., DSPE-PEG), including PEGylated versions of any of the above lipids. In some embodiments, the nanoparticles provided herein comprise DOTAP;

[0056] In some embodiments, the nanoparticles provided herein comprise oil. In some embodiments, the oil is in liquid phase. Non-limiting examples of oils that can be used include alpha-tocopherol, coconut oil, dihydroisosqualene (DHIS), farnesene, grape seed oil, lauroyl polyoxyl glyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E. In some embodiments, the nanoparticles provided herein comprise triglyceride. Exemplary triglycerides include, but are not limited to, capric triglyceride, caprylic triglyceride, caprylic and capric triglycerides, triglyceride esters, and myristate triglyceride.

[0057] In some embodiments, the nanoparticles provided herein comprise a liquid organic material and a solid inorganic material. In some embodiments, the nanoparticles provided herein comprise inorganic particles. In some embodiments, the inorganic particles are solid inorganic particles. In some embodiments, the nanoparticles provided herein comprise inorganic particles within a hydrophobic core.

[0058] In some embodiments, the nanoparticles provided herein comprise a metal. In some embodiments, the nanoparticles provided herein comprise a metal in a hydrophobic core. The metal can be, but is not limited to, a metal salt, such as a transition metal salt, a metal oxide, such as a transition metal oxide, a metal hydroxide, such as a transition metal hydroxide, a metal phosphate, such as a transition metal phosphate, or a metalloid (e.g., silicon and silicon-based compounds or alloys). In some embodiments, the nanoparticles provided herein comprise aluminum oxide (Al 2 O 3 ), aluminum oxyhydroxide, iron oxide (Fe 3 O 4 , Fe 2 O 3 , FeO, or a combination thereof), titanium dioxide, silicon dioxide (SiO 2), aluminum hydroxyphosphate (Al(OH) x (PO 4 ) y ), calcium phosphate (Ca 3 (PO 4 ) 2 ), calcium hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), iron gluconate, or iron sulfate. The inorganic particles may be formed from one or more of the same or different metals (any metal including transition metals). In some embodiments, the inorganic particles are transition metal oxides. In some embodiments, the transition metal is magnetite (Fe 3 O 4 ), Maghemite (y-Fe 2 O 3 ), wüstite (FeO), or hematite (alpha (α)-Fe 2 O 3 ). In some embodiments, the metal is aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or surfactant, such as oleic acid, oleylamine, SDS, TOPO, or DSPA, is used to coat the inorganic solid nanoparticles before being mixed with the liquid oil to form the hydrophobic core. In some embodiments, the metal can comprise a paramagnetic, superparamagnetic, ferrimagnetic, or ferromagnetic compound. In some embodiments, the metal can be superparamagnetic iron oxide (Fe 3 O 4 ).

[0059] In some embodiments, the nanoparticles provided herein comprise cationic lipid; oil and inorganic particles.In some embodiments, the nanoparticles provided herein comprise DOTAP; squalene and / or glyceryl trimyristate-dynasan; and iron oxide.In some embodiments, the nanoparticles provided herein further comprise surfactant.Thus, in some embodiments, the nanoparticles provided herein comprise cationic lipid; oil, inorganic particles, and surfactant.

[0060] A surfactant is a compound that reduces the surface tension between two liquids or between a liquid and a solid component of the nanoparticles provided herein. Surfactants can be hydrophobic, hydrophilic, or amphiphilic. In some embodiments, the nanoparticles provided herein include a hydrophobic surfactant. Exemplary hydrophobic surfactants that can be used include, but are not limited to, sorbitan monolaurate (SPAN® 20), sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate (SPAN® 65), sorbitan monooleate (SPAN® 80), and sorbitan trioleate (SPAN® 85). Suitable hydrophobic surfactants include those with a hydrophilic lipophilic balance (HLB) value of 10 or less, such as 5 or less, 1 to 5, or 4 to 5. For example, the hydrophobic surfactant can be a sorbitan ester with an HLB value of 1 to 5, or 4 to 5.

[0061] In some embodiments, the nanoparticles provided herein comprise hydrophilic surfactants, also referred to as emulsifiers. In some embodiments, the nanoparticles provided herein comprise polysorbates. Polysorbates are oily liquids derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Exemplary hydrophilic surfactants that can be used include, but are not limited to, polysorbates, such as TWEEN®, Kolliphor, Scattics, Alkest, or Canarcel; polyoxyethylene sorbitan esters (polysorbates); polysorbate 80 (polyoxyethylene sorbitan monooleate, or TWEEN® 80); polysorbate 60 (polyoxyethylene sorbitan monostearate, or TWEEN® 60); polysorbate 40 (polyoxyethylene sorbitan monopalmitate, or TWEEN® 40); and polysorbate 20 (polyoxyethylene sorbitan monolaurate, or TWEEN® 20). In one embodiment, the hydrophilic surfactant is polysorbate 80.

[0062] The nanoparticles provided herein comprise a hydrophobic core surrounded by a lipid membrane (e.g., a cationic lipid such as DOTAP). In some embodiments, the hydrophobic core comprises one or more inorganic particles; a phosphate-terminated lipid; and a surfactant.

[0063] The inorganic solid nanoparticles described herein can be surface modified prior to mixing with liquid oil. For example, if the surface of the inorganic solid nanoparticles is hydrophilic, the inorganic solid nanoparticles can be coated with hydrophobic molecules (or surfactants) to facilitate miscibility of the inorganic solid nanoparticles with the liquid oil in the "oil" phase of the nanoemulsion particle. In some embodiments, the inorganic particles are coated with capping ligands, phosphate-terminated lipids, and / or surfactants. In some embodiments, the hydrophobic core comprises a phosphate-terminated lipid. Exemplary phosphate-terminated lipids that can be used include, but are not limited to, trioctylphosphine oxide (TOPO) or distearylphosphatidic acid (DSPA). In some embodiments, the hydrophobic core comprises a surfactant, such as a phosphorous acid-terminated surfactant, a carboxylic acid-terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant. An exemplary carboxylic acid-terminated surfactant includes oleic acid. An exemplary amine-terminated surfactant includes oleylamine. In some embodiments, the surfactant is distearyl phosphatidic acid (DSPA), oleic acid, oleylamine, or sodium dodecyl sulfate (SDS). In some embodiments, the inorganic solid nanoparticles are metal oxides, such as iron oxide, and a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, is used to coat the inorganic solid nanoparticles prior to mixing the inorganic solid nanoparticles with the liquid oil to form the hydrophobic core.

[0064] In some embodiments, the hydrophobic core comprises one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle, optionally coated with a phosphate-terminated lipid, phosphorus-terminated surfactant, carboxylate-terminated surfactant, sulfate-terminated surfactant, or amine-terminated surfactant; and a naturally occurring or synthetic squalene-containing liquid oil; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.

[0065] In some embodiments, the hydrophobic core comprises one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles, optionally coated with TOPO, as well as a naturally occurring or synthetic squalene-containing liquid oil; the cationic lipid DOTAP; a hydrophobic surfactant including sorbitan monostearate; and a hydrophilic surfactant including polysorbate 80.

[0066] In some embodiments, the hydrophobic core is comprised of one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle, optionally coated with a phosphate-terminated lipid, phosphorus-terminated surfactant, carboxylate-terminated surfactant, sulfate-terminated surfactant, or amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid containing DOTAP; a hydrophobic surfactant containing a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant containing polysorbate. In some embodiments, the hydrophobic core is comprised of one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles, optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid DOTAP; a hydrophobic surfactant containing sorbitan monostearate; and a hydrophilic surfactant containing polysorbate 80. In some embodiments, the nanoparticles provided herein may comprise about 0.2% to about 40% w / v squalene, about 0.001% to about 10% w / v iron oxide nanoparticles, about 0.2% to about 10% w / v DOTAP, about 0.25% to about 5% w / v sorbitan monostearate, and about 0.5% to about 10% w / v polysorbate 80. In some embodiments, the nanoparticles provided herein may comprise about 2% to about 6% w / v squalene, about 0.01% to about 1% w / v iron oxide nanoparticles, about 0.2% to about 1% w / v DOTAP, about 0.25% to about 1% w / v sorbitan monostearate, and about 0.5% to about 5% w / v polysorbate 80. In some embodiments, the nanoparticles provided herein may comprise about 0.2% to about 40% w / v squalene, about 0.001% to about 10% w / v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, about 0.2% to about 10% w / v DOTAP, about 0.25% to about 5% w / v sorbitan monostearate, and about 0.5% to about 10% w / v polysorbate 80.In some embodiments, the nanoparticles provided herein may comprise about 2% to about 6% w / v squalene, about 0.01% to about 1% w / v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, about 0.2% to about 1% w / v DOTAP, about 0.25% to about 1% w / v sorbitan monostearate, and about 0.5% to about 5% w / v polysorbate 80.

[0067] In some embodiments, the compositions described herein comprise at least one nanoparticle formulation described in Table 1. In some embodiments, the compositions described herein comprise any one of NP-1 to NP-30. In some embodiments, the compositions described herein comprise any one of NP-1 to NP-31. In some embodiments, the nanoparticles provided herein are mixed with the nucleic acids provided herein. In some embodiments, the nanoparticles provided herein are made by homogenization and sonication techniques. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0068] In some embodiments, the nanoparticles provided herein comprise sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP, squalene, and are free of solid particles. In some embodiments, the nanoparticles provided herein comprise sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP, squalene, and iron oxide particles. In some embodiments, the nanoparticles provided herein comprise an immune stimulant. In some embodiments, the immune stimulant is squalene. In some embodiments, the immune stimulant is Miglyol 810 or Miglyol 812. Miglyol 810 is a triglyceride ester of saturated caprylic and capric fatty acids and glycerol. Miglyol 812 is a triglyceride ester of saturated coconut oil / palm kernel oil derived caprylic and capric fatty acids and vegetable derived glycerol. In some embodiments, the immune stimulant may reduce the total amount of protein produced but increase the immune response to the compositions provided herein (e.g., when delivered as a vaccine). In some embodiments, the immune stimulant may increase the total amount of protein produced but decrease the immune response to the compositions provided herein.

[0069] The nanoparticles provided herein can be of various average diameter sizes. In some embodiments, the nanoparticles provided herein have an average diameter (z-average hydrodynamic diameter, measured by dynamic light scattering) ranging from about 20 nanometers (nm) to about 200 nm. In some embodiments, the z-average diameter of the nanoparticles ranges from about 20 nm to about 150 nm, about 20 nm to about 100 nm, about 20 nm to about 80 nm, or about 20 nm to about 60 nm. In some embodiments, the z-average diameter of the nanoparticles ranges from about 40 nm to about 200 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 40 nm to about 90 nm, about 40 nm to about 80 nm, or about 40 nm to about 60 nm. In one embodiment, the z-average diameter of the nanoparticles is about 40 nm to about 80 nm. In some embodiments, the z-average diameter of the nanoparticles is about 40 nm to about 60 nm. In some embodiments, the nanoparticles are up to 100 nm in diameter. In some embodiments, the nanoparticles are 50-70 nm in diameter. In some embodiments, the nanoparticles are 40-80 nm in diameter. In some embodiments, the inorganic particles (e.g., iron oxide) within the hydrophobic core of the nanoparticles can have an average diameter (number weighted average diameter) ranging from about 3 nm to about 50 nm. For example, the inorganic particles can have an average diameter of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. In some embodiments, the ratio of ester and lipid results in a particle size of 30 nm to 200 nm. In some embodiments, the ratio of ester and lipid results in a particle size of 40 nm to 70 nm.

[0070] The nanoparticles provided herein may be characterized by their polydispersity index (PDI), which is an indication of their quality with respect to size distribution. In some embodiments, the average polydispersity index (PDI) of the nanoparticles provided herein ranges from about 0.1 to about 0.5. In some embodiments, the average PDI of the nanoparticles may range from about 0.2 to about 0.5, about 0.1 to about 0.4, about 0.2 to about 0.4, about 0.2 to about 0.3, or about 0.1 to about 0.3.

[0071] In some embodiments, the nanoparticles provided herein comprise a molar ratio of oil to surfactant ranging from about 0.1:1 to about 20:1, from about 0.5:1 to about 12:1, from about 0.5:1 to about 9:1, from about 0.5:1 to about 5:1, from about 0.5:1 to about 3:1, or from about 0.5:1 to about 1:1. In some embodiments, the nanoparticles provided herein comprise a ratio of hydrophilic surfactant to lipid ranging from about 0.1:1 to about 2:1, from about 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about 0.5:1 to about 1:1, or from about 0.6:1 to about 1:1. In some embodiments, the nanoparticles provided herein comprise a ratio of hydrophobic surfactant to lipid ranging from about 0.1:1 to about 5:1, from about 0.2:1 to about 3:1, from about 0.3:1 to about 2:1, from about 0.5:1 to about 2:1, or from about 1:1 to about 2:1.

[0072] In some embodiments, the nanoparticles provided herein comprise about 0.2% to about 40% w / v liquid oil, about 0.001% to about 10% w / v inorganic solid nanoparticles, about 0.2% to about 10% w / v lipid, about 0.25% to about 5% w / v hydrophobic surfactant, and about 0.5% to about 10% w / v hydrophilic surfactant. In some embodiments, the lipid comprises a cationic lipid, the oil comprises squalene, and / or the hydrophobic surfactant comprises a sorbitan ester.

[0073] In some embodiments, the nanoparticles provided herein comprise esters in a ratio that results in a hydrophilic-lipophilic balance of between 8 and 11.

[0074] In some embodiments, the nucleic acids provided herein are incorporated into, associated with, or complexed with the lipid carriers provided herein to form lipid carrier-nucleic acid complexes formed by non-covalent or reversible covalent interactions. nucleic acid

[0075] Compositions comprising nucleic acids are provided herein. In some embodiments, the nucleic acid is complexed with a nanoparticle. In some embodiments, the nucleic acid is complexed with a membrane of the nanoparticle. In some embodiments, the nucleic acid is complexed with a hydrophilic surface of the nanoparticle. In some embodiments, the nucleic acid is in the interior of the nanoparticle. In some embodiments, the nucleic acid is in the interior of the hydrophobic core.

[0076] In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The nucleic acid is linear or includes a secondary structure (e.g., a hairpin). In some embodiments, the nucleic acid is a polynucleotide that includes modified nucleotides or bases, and / or their analogs. The polynucleotide may include modified nucleotides, such as methylated nucleotides, and their analogs. Modifications to the nucleotide structure, if present, may have been made before or after assembly of the compositions provided herein. Modified nucleoside and nucleotide modified nucleobases that may be incorporated into and present in RNA molecules include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms 2m6A(2-methylthio-N6-methyladenosine);i6A(N6-isopentenyl adenosine);ms2i6A(2-methylthio-N6-isopentenyl adenosine);io6A(N6-(cis-hydroxyisopentenyl) adenosine);ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine);g6A(N6-glycinylcarbamoyl adenosine);t6A(N 6-Threonylcarbamoyl adenosine;ms2t6A(2-Methylthio-N6-threonylcarbamoyl adenosine);m6t6A(N6-Methyl-N6-threonylcarbamoyl adenosine);hn6A(N6-Hydroxynorvalylcarbamoyl adenosine);ms2hn6A(2-Methylthio-N6-Hydroxynorvalylcarbamoyl adenosine);Ar(p)(2'-O-Ribosyl adenosine (phosphate));I(inosine);m1I(1-methylinosine);m'Im(1,2'-O-dimethylinosine);m3C(3-methylcytidine);Cm(2T-O-methylcytidine);s2C(2-thiocytidine);ac4C(N4-acetylcytidine);f5C(5-phonylcytidine);m5Cm(5,2-O-dimethylcytidine);ac4Cm(N4-acetyl2T-O-methylcytidine);k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (incompletely modified (underm odified)hydroxywybutosine;imG(wybutosine);mimG(methylguanosine);Q(queuosine);oQ(epoxyqueuosine);galQ(galtactosyl-queuosine);manQ(mannosyl-queuosine);preQo(7-cyano-7-deazaguanosine);preQi(7-aminomethyl-7-deazaguanosine);G*(archaeosine);D(dihydrouridine);m5Um(5,2'-O-dimethyluridine);s4U(4-thiouridine);m5s2U(5-methyl-2-thiouridine);s2 Um(2-thio-2'-O-methyluridine);acp3U(3-(3-amino-3-carboxypropyl)uridine);hoSU(5-hydroxyuridine);moSU(5-methoxyuridine);cmo5U(uridine-5-oxyacetic acid);mcmo5U(uridine-5-oxyacetic acid methyl ester);chm5U(5-(carboxyhydroxymethyl)uridine);mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm5U(5-methoxycarbonylmethyluridine);mcm5Um(S-methoxycarbonylmethyluridine); nylmethyl-2-O-methyluridine; mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine);cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,TO-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethyl methylguanosine);m3Um(3,2T-O-dimethyluridine);m5D(5-methyldihydrouridine);f5Cm(5-formyl-2'-O-methylcytidine);m1Gm(1,2'-O-dimethylguanosine);m'Am(1,2-O-dimethyladenosine)irinomethyluridine;tm5s2U(S-taurinomethyl-2-thiouridine));imG-14(4-demethylguanosine);imG2(isoguanosine);ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C; 1 ~C 6 )-Alkyluracil, 5-methyluracil, 5-(C 2 ~C 6 )-Alkenyluracil, 5-(C 2 ~C 6 )-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C 1 ~C 6 )-Alkylcytosine, 5-methylcytosine, 5-(C 2 ~C 6 )-alkenylcytosine, 5-(C 2 ~C 6 )-Alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N 2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Any one or any combination of these modified nucleobases can be included in the self-replicating RNA of the present invention. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the nucleic acid may contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. For example, the RNA sequence can be modified with respect to its codon usage to increase the translational efficiency and half-life of the RNA. To increase the half-life of the RNA, a polyA tail (e.g., with about 30 or more adenosine residues) can be attached to the 3' end of the RNA. The 5' end of the RNA can be capped with a modified ribonucleotide or derivative having the structure m7G(5')ppp(5')N (cap 0 structure), which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using vaccinia virus capping enzyme (VCE), which consists of mRNA triphosphatase, guanylyl-transferase, and guanine-7-methyltransferase, which catalyzes the construction of the N7-monomethylated cap 0 structure). The cap structure can provide stability and translational efficiency to the RNA molecule. The 5' cap of an RNA molecule can be further modified by 2'-O-methyltransferase, resulting in the generation of the Cap 1 structure (m7Gppp[m2'-O]N), which can further increase translational efficiency and may also increase potency in vivo.

[0077] In some embodiments, the compositions provided herein comprise one or more nucleic acids. In some embodiments, the compositions provided herein comprise two or more nucleic acids. In some embodiments, the compositions provided herein comprise at least one DNA. In some embodiments, the compositions provided herein comprise at least one RNA. In some embodiments, the compositions provided herein comprise at least one DNA and at least one RNA. In some embodiments, the nucleic acids provided herein are present in an amount of more than 5 ng to about 1 mg. In some embodiments, the nucleic acids provided herein are present in an amount of up to about 25 ng, 50 ng, 75 ng, 100 ng, 150 ng, 175 ng. In some embodiments, the nucleic acids provided herein are present in an amount of up to about 1 mg. In some embodiments, the nucleic acids provided herein are present in an amount of about 0.05 μg, 0.1 μg, 0.2 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 12.5 μg, 15 μg, 25 μg, 40 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg. In some embodiments, the nucleic acids provided herein are present in an amount of 0.05 μg, 0.1 μg, 0.2 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 12.5 μg, 15 μg, 25 μg, 40 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1 mg. In some embodiments, the nucleic acid is at least about 200, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 nucleotides in length.In some embodiments, the nucleic acid is up to about 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 nucleotides in length. In some embodiments, the nucleic acid is about 7500, 10,000, 15,000, or 20,000 nucleotides in length.

[0078] In some embodiments, the compositions provided herein comprise at least one nucleic acid sequence comprising a sequence encoding a viral antigen. In some embodiments, the viral antigen is a surface protein or a transmembrane protein. In some embodiments, the viral antigen is a spike protein, a glycoprotein, or an envelope protein. In some embodiments, the viral antigen is a coronavirus antigen or a fragment thereof. In some embodiments, the antigen is a SARS-CoV antigen, a SARS-CoV-2 antigen, a Middle East Respiratory Syndrome (MERS) coronavirus antigen, a fragment thereof, or a variant thereof. SARS-CoV-2 spike protein antigen

[0079] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that causes an infectious disease called COVID-19 (coronavirus disease 2019). SARS-CoV-2 virus variants include alpha, beta, delta, mu, and omicron variants. The alpha variant (B.1.1.7), beta variants (B.1.351, B.1.351.2, B.1.351.3), delta variants (B.1.617.2, AY.1, AY.2, AY.3), omicron variant (B.1.1.529), and gamma variants (P.1, P.1.1, P.1.2), which are circulating in the United States, are classified as variants of concern. The SARS-CoV-2 structure, its components along with the variants and their various features are described below.

[0080] Coronaviruses are enveloped, single-stranded RNA viruses with four structural proteins known as S (spike) protein, E (envelope) protein, M (membrane) protein, and N (nucleocapsid) protein. The N protein carries the RNA genome, while the S, E, and M proteins together create the viral envelope. For SARS-CoV-2, the spike (S) protein facilitates the attachment and fusion of the virus with the host cell membrane via the host cell receptor, angiotensin-converting enzyme 2 (ACE2). The S protein mediates the entry of the virus into the host cell. The total length of the SARS-CoV-2 spike is generally 1273 amino acids, including a signal peptide (amino acids 1–13) located at the N-terminus, an S1 subunit (residues 14–685), and an S2 subunit (residues 686–1273). The last two regions are responsible for receptor binding and membrane fusion, respectively. Within the S1 subunit, there is an N-terminal domain (residues 14–305) and a receptor binding domain (RBD, residues 319–541). The S2 subunit contains the fusion peptide (FP) (residues 788–806), the heptapeptide repeat sequence 1 (HR1) (residues 912–984), HR2 (residues 1163–1213), the TM domain (residues 1213–1237), and the cytoplasmic domain (residues 1237–1273). Specifically, HR1 and HR2 are composed of repeats of the heptapeptide: HPPHCPC, where H is a hydrophobic or conventionally bulky residue, P is a polar or hydrophilic residue, and C is another charged residue. HR1 and HR2 form a six-helix bundle (6-HB) that is referred to as the "stem-helix" domain of the S2 protein. When the RBD binds to ACE2 on the host cell membrane, the insertion of the FP into the target cell membrane changes the conformation of S2, exposing the pre-hairpin coiled coil of the HR1 domain and inducing the interaction of the HR2 domain with the HR1 trimer to form a stem helix (6-HB), thus bringing the viral envelope and the cell membrane into close proximity for viral fusion and entry. The amino acid sequence of the wild-type spike protein is presented in SEQ ID NO: 17.The amino acid sequence corresponding to the stem helix is ​​presented in SEQ ID NO:18.

[0081] Coronaviruses periodically undergo antigenic drift, a type of genetic variation in the virus that results from the accumulation of mutations in the viral genes that code for the viral surface proteins that are recognized by host antibodies. Several SARS-CoV-2 variants have been identified by public health officials and are presented below in Table 2. [Table 2-1] [Table 2-2] [Table 2-3]

[0082] Compositions comprising coronavirus protein antigens are provided herein. In some embodiments, the coronavirus protein antigens provided herein are derived from variants of concern (VOCs) of SARS-CoV-2. Variants of concern are variants for which there is evidence of increased infection / transmissibility, more severe disease (e.g., increased hospitalization or death), significantly reduced neutralization by antibodies generated during prior infection or with vaccination, reduced efficacy of treatment or vaccines, or failure to be diagnostically detected. Potential attributes of variants of concern include, in addition to potential attributes of variants of interest, (i) evidence of an impact on diagnosis, treatment, or vaccines, (ii) widespread interference with diagnostic test targets, (iii) evidence of substantially reduced susceptibility to one or more classes of therapeutics, (iv) evidence of significantly reduced neutralization by antibodies generated during prior infection or with vaccination, (v) evidence of reduced vaccine-induced protection from severe disease, (vi) evidence of increased infection / transmissibility, and (vii) evidence of increased disease severity.

[0083] In some embodiments, the coronavirus protein antigens provided herein are derived from SARS-CoV-2 variants of interest (VOI). Variants of interest are variants that have specific genetic markers associated with altered receptor binding, reduced neutralization by antibodies generated in response to prior infection or vaccination, reduced efficacy of treatment, potential impact on diagnosis, or prediction of increased infection / transmissibility or disease severity. Potential attributes of variants of interest include (i) specific genetic markers predicted to affect transmission, diagnosis, therapeutics, or immune escape, (ii) evidence that they are responsible for a high proportion of cases or unique outbreak clusters, and (iii) limited prevalence or spread in the United States or other countries.

[0084] Compositions are provided herein that include a nucleic acid encoding a SARS-CoV-2 spike protein antigen. In some embodiments, the SARS-CoV-2 spike protein antigen is derived from a variant of SARS-CoV-2 listed in Table 2. In some embodiments, the SARS-CoV-2 spike protein antigen comprises one or more mutations or amino acid substitutions listed in Table 2. In some embodiments, the SARS-CoV-2 spike protein antigen comprises one or more mutations or amino acid substitutions in the receptor binding domain (RBD). In some embodiments, the SARS-CoV-2 spike protein antigen comprises one or more mutations or amino acid substitutions in the HR1 domain or the HR2 domain. In some embodiments, the SARS-CoV-2 spike protein antigen comprises a glycine residue at position 614 of the surface glycoprotein. In some embodiments, the SARS-CoV-2 spike protein antigen is derived from an alpha variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is derived from a beta variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from a delta variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from a gamma variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from an iota variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from a kappa variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from a lambda variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from a mu variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein antigen is from an omicron variant of SARS-CoV-2.

[0085] In some embodiments, the nucleic acids provided herein encode an antigen or fragment thereof listed in Table 3. In some embodiments, the nucleic acids comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence that specifically binds to an antigen listed in Table 3. In some embodiments, the nucleic acids provided herein comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an RNA sequence listed in Table 3. In some embodiments, the nucleic acids provided herein comprise a DNA sequence that is complementary to an RNA sequence listed in Table 3. The percent (%) sequence identity of a given sequence to a reference sequence is defined as the percentage of identical residues identified after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Percent identity can be calculated using alignment methods known in the art, for example, publicly available software such as BLAST, Align, ClustalW2, etc., can be used to align sequences. Those skilled in the art can determine appropriate parameters for alignment, but the default parameters of BLAST are specifically contemplated. Exemplary nucleic acid sequences encoding exemplary SARS-CoV-2 antigens are listed in Table 3. [Table 3]

[0086] In some embodiments, the compositions provided herein comprise a SARS-CoV-2 spike protein or a fragment thereof, wherein the SARS-CoV-2 spike protein amino acid sequence is encoded by any of the nucleic acid sequences disclosed herein. In some embodiments, the compositions provided herein comprise an RNA sequence set forth in any one of SEQ ID NOs: 1-8. In some embodiments, the spike protein has an amino acid sequence corresponding to any of the sequences set forth in any one of SEQ ID NOs: 9-19.

[0087] In some embodiments, the compositions provided herein comprise (i) an optional vector backbone, and (ii) a nucleic acid sequence comprising any of the antigen sequences set forth in SEQ ID NOs:24-31, or any nucleic acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 97% identical to any of the antigen sequences set forth in SEQ ID NOs:24-31.

[0088] In some embodiments, the compositions provided herein comprise (i) a vector backbone set forth in one of SEQ ID NOs: 32-38, and (ii) a nucleic acid sequence comprising any sequence encoding an amino acid sequence defining any SARS-CoV-2 spike protein, or any sequence encoding an amino acid sequence at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 97% identical to a sequence defining any SARS-CoV-2 spike protein. In some embodiments, the compositions provided herein comprise a DNA sequence complementary to any one of SEQ ID NOs: 24-38.

[0089] In some embodiments, the compositions provided herein comprise a nucleic acid sequence comprising: (i) a vector backbone set forth in one of SEQ ID NOs: 32-38, or any sequence at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 97% identical to a sequence set forth in one of SEQ ID NOs: 32-38; and (ii) any sequence encoding an amino acid sequence defining any SARS-CoV-2 spike protein, or any sequence encoding an amino acid sequence at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 97% identical to a sequence defining any SARS-CoV-2 spike protein. self-replicating nucleic acids

[0090] Compositions comprising self-replicating nucleic acids are provided herein. The SARS-CoV-2 spike antigen or fragment thereof provided herein may be encoded as part of a self-replicating nucleic acid construct. In some embodiments, the self-replicating nucleic acid molecule comprises at least one or more genes selected from the group consisting of viral replicase, viral protease, viral helicase and other nonstructural viral proteins, and also comprises a 5'-terminal cis-acting replication sequence, a 3'-terminal cis-acting replication sequence, and an antigenic sequence encoding the SARS-CoV-2 spike protein. A subgenomic promoter directing the expression of the heterologous sequence(s) can be included in the self-replicating nucleotide sequence. If desired, the heterologous sequence can be fused in frame with other coding regions in the self-replicating RNA and / or can be under the control of an internal ribosome entry site (IRES).

[0091] In some embodiments, the self-replicating nucleotide sequence is a self-replicating RNA molecule. The self-replicating RNA molecule is designed so that it cannot induce the production of infectious virus particles. This can be achieved, for example, by removing one or more viral genes in the self-replicating RNA that code for structural proteins required for the production of virus particles. For example, when the self-replicating RNA molecule is based on an alphavirus, such as Sindbis virus (SIN), Semliki Forest virus and Venezuelan equine encephalitis virus (VEE), one or more genes that code for viral structural proteins, such as capsid and / or envelope glycoproteins, can be removed. If desired, the self-replicating RNA molecule of the present invention is designed to induce the production of attenuated or virulent infectious virus particles, or to produce virus particles that can be infected once after.

[0092] When delivered to an animal cell, a self-replicating RNA molecule, even in the absence of any proteins, can result in the production of multiple daughter RNAs by transcription from itself (or from an antisense copy of itself). The self-replicating RNA can be directly translated after delivery to a cell, which translation leads to an RNA-dependent RNA polymerase that in turn produces transcripts of the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These transcripts are antisense to the delivered RNA and can themselves be translated to result in the in situ expression of the encoded SARS-CoV-2 spike protein, or can be transcribed into additional transcripts of the same sense as the delivered RNA, which are translated to result in the in situ expression of the encoded SARS-CoV-2 spike protein(s).

[0093] The self-replicating RNA molecules provided herein may contain one or more modified nucleotides, thus improving stability and resisting degradation and clearance in vivo, as well as other advantages. In some embodiments, the self-replicating RNA molecules containing modified nucleotides avoid or reduce stimulation of endosomal and cytoplasmic immune receptors when the self-replicating RNA is delivered into cells. This allows for self-replication, amplification, and expression of proteins. In addition, the self-replicating RNA containing modified nucleotides reduces activation of the innate immune system and subsequent undesirable consequences (e.g., inflammation at the injection site, irritation at the injection site, pain, etc.), thus reducing safety concerns compared to self-replicating RNAs that do not contain modified nucleotides. The RNA molecules produced as a result of self-replication are recognized as foreign nucleic acids by cytoplasmic immune receptors. Thus, the self-replicating RNA molecules containing modified nucleotides provide efficient amplification of RNA and expression of SARS-CoV-2 spike protein in host cells, as well as adjuvant effects.

[0094] In some embodiments, the self-replicating RNA molecules provided herein contain at least one modified nucleotide. Modified nucleotides that are not part of the 5' cap can be used (e.g., in addition to modifications that are part of the 5" cap). Thus, a self-replicating RNA molecule can contain a modified nucleotide at a single position, can contain a particular modified nucleotide (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine) at two or more positions, or can contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides (e.g., at each of one or more positions). Preferably, the self-replicating RNA molecule comprises modified nucleotides that contain modifications on or within the nitrogenous base, but not modified sugar or phosphate moieties. In some examples, between 0.001% and 99% or 100% of the nucleotides in a self-replicating RNA molecule are modified nucleotides. For example, a self-replicating RNA molecule can be 0.001%-25%, 0.01%-25%, 0.1%-25%, or 1%-25% of the nucleotides in the RNA molecule are modified nucleotides. In other examples, between 0.001% and 99% or 100% of the unmodified specific nucleotides in the self-replicating RNA molecule are replaced with modified nucleotides. For example, about 1% of the nucleotides in a self-replicating RNA molecule that contain uridine can be modified, for example, by replacing the uridine with pseudouridine. In other examples, a desired amount (percentage) of two, three, or four specific nucleotides (nucleotides that contain uridine, cytidine, guanosine, or adenine) in the self-replicating RNA molecule are modified nucleotides. For example, 0.001%-25%, 0.01%-25%, 0.1%-25%, or 1%-25% of the nucleotides in a particular self-replicating RNA molecule are modified nucleotides.In other examples, 0.001% to 20%, 0.001% to 15%, 0.001% to 10%, 0.01% to 20%, 0.01% to 15%, 0.1% to 25%, 0.01% to 10%, 1% to 20%, 1% to 15%, 1% to 10%, or about 5%, about 10%, about 15%, about 20% of the nucleotides in a particular self-replicating RNA molecule are modified nucleotides. It is preferred that less than 100% of the nucleotides in a particular self-replicating RNA molecule are modified nucleotides. It is also preferred that less than 100% of the nucleotides in a particular self-replicating RNA molecule are modified nucleotides. Thus, preferred self-replicating RNA molecules contain at least some unmodified nucleotides.

[0095] Self-replicating RNA molecules containing at least one modified nucleotide can be prepared using any suitable method. Several suitable methods for making RNA molecules containing modified nucleotides are known in the art. For example, self-replicating RNA molecules containing modified nucleotides can be prepared by transcribing (e.g., in vitro transcription) DNA encoding the self-replicating RNA molecule using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, etc., or mutants of these polymerases that allow efficient incorporation of modified nucleotides into RNA molecules. The transcription reaction contains nucleotides and modified nucleotides, as well as other components that support the selected polymerase activity, such as suitable buffers and suitable salts. The incorporation of nucleotide analogs into self-replicating RNA can be engineered, for example, to alter the stability of such RNA molecules, to increase resistance to RNases, to establish replication (the "infectivity" of RNA) after introduction into a suitable host cell, and / or to induce or reduce innate and adaptive immune responses. Suitable synthetic methods can be used alone or in combination with one or more other methods (e.g., recombinant DNA or RNA techniques) to generate self-replicating RNA molecules containing one or more modified nucleotides.

[0096] Nucleic acid synthesis can also be carried out using suitable recombinant methods well known and conventional in the art, including cloning, processing, and / or expression of polynucleotides and gene products encoded by such polynucleotides.DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic polynucleotides are examples of known techniques that can be used to design and engineer polynucleotide sequences.Site-directed mutagenesis can be used to modify nucleic acids and encoded proteins, for example, to insert new restriction sites, change glycosylation patterns, alter codon preferences, generate splice variants, introduce mutations, and the like.

[0097] In some embodiments, the nucleic acid provided herein encodes an RNA polymerase. In some embodiments, the nucleic acid provided herein encodes a viral RNA polymerase. In some embodiments, the nucleic acid provided herein encodes (1) a viral RNA polymerase, and (2) a protein or a functional fragment thereof. In some embodiments, the composition provided herein comprises a first nucleic acid that encodes a viral RNA polymerase, and a second nucleic acid that encodes a protein or a functional fragment thereof.

[0098] The composition provided herein comprises self-replicating RNA.Self-replicating RNA (also called replicon) includes any genetic element that can replicate mainly under its own control, such as plasmid, cosmid, bacmid, phage or virus.Self-replication provides a system for self-amplification of the nucleic acid provided herein in mammalian cells.In some embodiments, the self-replicating RNA is single-stranded.In some embodiments, the self-replicating RNA is double-stranded.

[0099] The RNA polymerases provided herein may include, but are not limited to, alphavirus RNA polymerase, Eastern Equine Encephalitis Virus (EEEV) RNA polymerase, Seibu Equine Encephalitis Virus (WEEV), Venezuelan Equine Encephalitis Virus (VEEV), Chikungunya Virus (CHIKV), Semliki Forest Virus (SFV), or Sindbis Virus (SINV). In some embodiments, the RNA polymerase is VEEV RNA polymerase. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 85% identity to the nucleic acid sequence of SEQ ID NO:20. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 90% identity to the nucleic acid sequence of SEQ ID NO:20. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 95% identity to the nucleic acid sequence of SEQ ID NO:20. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 99% identity to the nucleic acid sequence of SEQ ID NO:20. In some embodiments, the nucleic acid encoding the RNA polymerase is SEQ ID NO:20.

[0100] In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 85% identity to RELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGP (SEQ ID NO:21), TQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTE (SEQ ID NO:22), or SEQ ID NO:23. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 90% identity to SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 95% identity to SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 99% identity to SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In some embodiments, the amino acid sequence of the VEEV RNA polymerase is SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.

[0101] Compositions and methods are provided herein that include a replicon RNA (repRNA) that encodes one or more structural proteins from a non-enveloped virus. In some embodiments, the repRNA encodes a protease. In some embodiments, the repRNA encodes a 3CD protease. In some embodiments, the structural proteins and the protease are co-expressed. In further embodiments, the repRNA comprises one or more open reading frames. In some embodiments, the open reading frames are separated by an internal ribosome entry site (IRES). In some embodiments, the open reading frames are separated by a ribosome skipping peptide sequence. In some embodiments, the ribosome skipping peptide sequence is derived from Thosea asigna virus (T2A). Combination Compositions

[0102] Provided herein is a composition comprising a nanoparticle as described herein and a nucleic acid encoding a coronavirus antigen. Further provided herein is a nanoemulsion comprising a plurality of nanoparticles as provided herein. In some embodiments, the nucleic acid encodes a self-replicating RNA polymerase. In some embodiments, the nucleic acid encodes a self-replicating RNA-dependent RNA polymerase. In some embodiments, the nucleic acid further encodes an RNA polymerase. In some embodiments, the RNA polymerase is a viral RNA polymerase. In some embodiments, the nucleic acid encoding the RNA polymerase is on the same nucleic acid strand as the nucleic acid sequence encoding the protein (e.g., cis). In some embodiments, the nucleic acid encoding the RNA polymerase is on a different nucleic acid strand than the nucleic acid sequence encoding the protein (e.g., trans). In some embodiments, the nucleic acid encoding the RNA polymerase is a DNA molecule. In some embodiments, the nucleic acid sequence encoding the antigen provided herein is a DNA molecule or an RNA molecule. In some embodiments, the antigen provided herein is encoded by DNA. Nanoparticles for encapsulation include, without limitation, one of NP-1 to NP-30, or any one of NP-1 to NP-31. In some embodiments, the nanoparticles include NP-1. In some embodiments, the nanoparticles comprise NP-2. In some embodiments, the nanoparticles comprise NP-3. In some embodiments, the nanoparticles comprise NP-4. In some embodiments, the nanoparticles comprise NP-5. In some embodiments, the nanoparticles comprise NP-6. In some embodiments, the nanoparticles comprise NP-7. In some embodiments, the nanoparticles comprise NP-8. In some embodiments, the nanoparticles comprise NP-9. In some embodiments, the nanoparticles comprise NP-10. In some embodiments, the nanoparticles comprise NP-11. In some embodiments, the nanoparticles comprise NP-12. In some embodiments, the nanoparticles comprise NP-13. In some embodiments, the nanoparticles comprise NP-14. In some embodiments, the nanoparticles comprise NP-15. In some embodiments, the nanoparticles comprise NP-16. In some embodiments, the nanoparticles comprise NP-17.In some embodiments, the nanoparticles comprise NP-18. In some embodiments, the nanoparticles comprise NP-18. In some embodiments, the nanoparticles comprise NP-19. In some embodiments, the nanoparticles comprise NP-20. In some embodiments, the nanoparticles comprise NP-21. In some embodiments, the nanoparticles comprise NP-22. In some embodiments, the nanoparticles comprise NP-23. In some embodiments, the nanoparticles comprise NP-24. In some embodiments, the nanoparticles comprise NP-25. In some embodiments, the nanoparticles comprise NP-26. In some embodiments, the nanoparticles comprise NP-27. In some embodiments, the nanoparticles comprise NP-28. In some embodiments, the nanoparticles comprise NP-28. In some embodiments, the nanoparticles comprise NP-29. In some embodiments, the nanoparticles comprise NP-30. In some embodiments, the nanoparticles comprise NP-31. In some embodiments, the nanoparticles comprise any of NP-1-NP-31 and a cryoprotectant. In some embodiments, the cryoprotectant is a sugar as described herein. Nucleic acids for encapsulation include, without limitation, a region comprising any one or more of SEQ ID NOs: 1-8 and / or 24-31. In some cases, the nucleic acid further comprises a region encoding an RNA polymerase, e.g., a region comprising the sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 2, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 3, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 4, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 5, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 6, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 7, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 8, or a functional fragment thereof.In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:24, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:25, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:26, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:27, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:28, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:29, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:30, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:31, or a functional fragment thereof. In some embodiments, the nucleic acid described herein comprises a sequence that encodes an infectious disease antigen described herein and a sequence that encodes an RNA-dependent RNA polymerase. In some embodiments, the RNA-dependent RNA polymerase is a VEEV RNA polymerase. In some embodiments, the two nucleic acids encoding the elements are in separate nucleic acids. In some embodiments, the two nucleic acids encoding the elements are present on the same nucleic acid.

[0103] In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:2 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:3 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:4 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:5 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:6 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:7 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:8 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-1 and a nucleic acid encoding a SARS-CoV-2 spike protein antigen comprising a glycine at position 614 of the amino acid sequence (G614). In some embodiments, the compositions provided herein comprise NP-1 and a nucleic acid encoding a SARS-CoV-2 spike protein antigen, wherein the SARS-CoV-2 spike protein antigen comprises a mutation or amino acid substitution compared to SEQ ID NO:17. In some embodiments, the compositions provided herein comprise NP-1 and a nucleic acid comprising (i) SEQ ID NO:20, and (ii) a region encoding a SARS-CoV-2 spike protein antigen, wherein the SARS-CoV-2 spike protein antigen comprises a mutation or amino acid substitution compared to SEQ ID NO:17.In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:24 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:25 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:26 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:27 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:28 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:29 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:30 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-1 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO: 31, or a functional fragment thereof.

[0104] In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:2 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:3 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:4 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:5 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:6 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:7 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:8 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-2 and a nucleic acid encoding a SARS-CoV-2 spike protein antigen comprising a glycine at position 614 of the amino acid sequence (G614). In some embodiments, the compositions provided herein comprise NP-2 and a nucleic acid encoding a SARS-CoV-2 spike protein antigen, wherein the SARS-CoV-2 spike protein antigen comprises a mutation or amino acid substitution compared to SEQ ID NO:17. In some embodiments, the compositions provided herein comprise NP-2 and a nucleic acid comprising (i) SEQ ID NO:20, and (ii) a region encoding a SARS-CoV-2 spike protein antigen, wherein the SARS-CoV-2 spike protein comprises a mutation or amino acid substitution compared to SEQ ID NO:17.In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:24 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:25 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:26 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:27 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:28 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:29 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:30 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-2 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO: 31, or a functional fragment thereof.

[0105] In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:2 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:3 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:4 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:5 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:6 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:7 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:8 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-30 and a nucleic acid encoding a SARS-CoV-2 spike protein antigen comprising a glycine at position 614 of the amino acid sequence (G614). In some embodiments, the compositions provided herein comprise NP-30 and a nucleic acid encoding a SARS-CoV-2 spike protein antigen, wherein the SARS-CoV-2 spike protein antigen comprises a mutation or amino acid substitution compared to SEQ ID NO:17. In some embodiments, the compositions provided herein comprise NP-30 and a nucleic acid comprising (i) SEQ ID NO:20, and (ii) a region encoding a SARS-CoV-2 spike protein antigen, wherein the SARS-CoV-2 spike protein antigen comprises a mutation or amino acid substitution compared to SEQ ID NO:17.In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:24 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:25 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:26 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:27 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:28 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:29 or a functional fragment thereof. In some embodiments, the compositions provided herein include NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:30 or a functional fragment thereof. In some embodiments, the compositions provided herein comprise NP-30 and a nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:31, or a functional fragment thereof.

[0106] The compositions provided herein can be characterized by the molar ratio of nitrogen:phosphate (N:P). The N:P ratio is determined by the amount of cationic lipid in the nitrogen-containing nanoparticle and the amount of nucleic acid used in the composition containing negatively charged phosphate. The molar ratio of lipid carrier to nucleic acid can be selected to increase the delivery efficiency of nucleic acid, increase the ability of the nucleic acid-carrying nanoemulsion composition to induce an immune response against an antigen, or increase the ability of the nucleic acid-carrying nanoemulsion composition to induce the production of antibody titers against an antigen in a subject. In some embodiments, the compositions provided herein have a molar ratio of lipid carrier to nucleic acid, which may be characterized by the molar ratio of nitrogen to phosphate, which may range from about 0.01:1 to about 1000:1, e.g., from about 0.2:1 to about 500:1, from about 0.5:1 to about 150:1, from about 1:1 to about 150:1, from about 1:1 to about 125:1, from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 1:1 to about 50:1, from about 5:1 to about 50:1, from about 5:1 to about 25:1, or from about 10:1 to about 20:1. In certain embodiments, the molar ratio of lipid carrier to nucleic acid, characterized by the molar ratio of nitrogen to phosphate (N:P), ranges from about 1:1 to about 150:1, from about 5:1 to about 25:1, or from about 10:1 to about 20:1. In one embodiment, the nanoemulsion composition has an N:P molar ratio of about 15: 1. In some embodiments, the nanoparticles comprise a nucleic acid provided herein covalently attached to the membrane.

[0107] The compositions provided herein may be characterized by the molar ratio of oil to surfactant. In some embodiments, the ratio of oil to surfactant is the molar ratio of squalene:DOTAP, hydrophobic surfactant, and hydrophilic surfactant. In some embodiments, the ratio of oil to surfactant is the molar ratio of squalene:DOTAP, sorbitan monostearate, and polysorbate 80. In some embodiments, the molar ratio of oil to surfactant ranges from about 0.1:1 to about 20:1, from about 0.5:1 to about 12:1, from about 0.5:1 to about 9:1, from about 0.5:1 to about 5:1, from about 0.5:1 to about 3:1, or from about 0.5:1 to about 1:1. In some embodiments, the molar ratio of oil to surfactant is at least about 0.1:1, at least about 0.2:1, at least about 0.3:1, at least about 0.4:1, at least about 0.5:1, at least about 0.6:1, or at least about 0.7:1. In some embodiments, the molar ratio of oil to surfactant is at least about 0.4:1 to at most 1:1.

[0108] The compositions provided herein may be characterized by the ratio of hydrophilic surfactant to lipid (e.g., cationic lipid). In some embodiments, the ratio of hydrophilic surfactant to lipid ranges from about 0.1:1 to about 2:1, from about 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about 0.5:1 to about 1:1, or from about 0.6:1 to about 1:1. The compositions provided herein may be characterized by the ratio of hydrophobic surfactant to lipid (e.g., cationic lipid). In some embodiments, the ratio of hydrophobic surfactant to lipid ranges from about 0.1:1 to about 5:1, from about 0.2:1 to about 3:1, from about 0.3:1 to about 2:1, from about 0.5:1 to about 2:1, or from about 1:1 to about 2:1.

[0109] Provided herein is a dry composition comprising a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid, an immunostimulant, and an RNA.Further provided herein is a dry composition comprising a sorbitan monostearate (e.g., SPAN® 60), a polysorbate 80 (e.g., TWEEN® 80), a DOTAP, an immunostimulant, and an RNA. Heat-stable SARS-COV-2 RNA vaccine, dried SARS-COV-2 RNA vaccine, and freeze-dried SARS-COV-2 RNA vaccine

[0110] Provided herein is a dry or lyophilized composition and a vaccine.Further provided herein is a pharmaceutical composition comprising the dry or lyophilized composition provided herein reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier.In some embodiments, the diluent is aqueous.In some embodiments, the diluent is water.

[0111] Lyophilized compositions are produced by a low-temperature dehydration process that involves freezing the composition, then reducing pressure and removing ice by sublimation. In certain cases, lyophilization also involves removing bound water molecules by a desorption process. In some embodiments, the compositions and vaccine compositions provided herein are spray-dried. Spray-drying is a process in which a solution is passed through an atomizer to produce a spray that is then exposed to a heated gas flow to promote rapid evaporation. Once a sufficient mass of liquid has evaporated, the solid material in the remaining droplets forms particles that are then separated from the gas flow (e.g., using a filter or cyclone). Drying helps the compositions and vaccine compositions provided herein to be stored at high temperatures (e.g., above 4 degrees Celsius) compared to the subzero temperatures required for storage of existing mRNA vaccines. In some embodiments, the dry composition and lyophilized composition provided herein comprises: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising: (i) a hydrophobic core; (ii) one or more inorganic nanoparticles; (iii) and one or more lipids; (b) one or more nucleic acids; and (c) at least one cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof. Additional examples of cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, 3-O-methyl-D-glucopyranose (3-OMG), polyethylene glycol (PEG), 1,2-propanediol, acetamide, trehalose, formamide, sugar, protein, and carbohydrate.

[0112] In some embodiments, the compositions and methods provided herein include at least one cryoprotectant. Exemplary cryoprotectants for inclusion are, but are not limited to, sucrose, maltose, trehalose, mannitol, or glucose, and any combination thereof. In some embodiments, additional or alternative cryoprotectants for inclusion are sorbitol, ribitol, erythritol, threitol, ethylene glycol, or fructose. In some embodiments, additional or alternative cryoprotectants for inclusion are dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, 3-O-methyl-D-glucopyranose (3-OMG), polyethylene glycol (PEG), 1,2-propanediol, acetamide, trehalose, formamide, sugars, proteins, and carbohydrates. In some embodiments, the cryoprotectant is present at about 1% w / v to about 20% w / v, preferably about 10% w / v to about 20% w / v, more preferably about 10% w / v. In certain aspects of the present disclosure, the cryoprotectant is sucrose. In some aspects of the present disclosure, the cryoprotectant is maltose. In some aspects of the present disclosure, the cryoprotectant is trehalose. In some aspects of the present disclosure, the cryoprotectant is mannitol. In some aspects of the present disclosure, the cryoprotectant is glucose. In some embodiments, the cryoprotectant is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 mg or more. In some embodiments, the cryoprotectant is present in an amount of about 50 to about 500 mg. In some embodiments, the cryoprotectant is present in an amount of about 200 to about 300 mg. In some embodiments, the cryoprotectant is present in an amount of about 250 mg. In some embodiments, the cryoprotectant is present in an amount of at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent by weight of the lyophilized composition.In some embodiments, the cryoprotectant is present in an amount of about 95% by weight of the lyophilized composition. In some embodiments, the cryoprotectant is present in an amount of 80-98%, 85-98%, 90-98%, or 94-96% by weight of the lyophilized composition. In some embodiments, the cryoprotectant is a sugar. In some embodiments, the sugar is sucrose, maltose, trehalose, mannitol, or glucose. In some embodiments, the sugar is sucrose. In some embodiments, sucrose is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 mg or more. In some embodiments, sucrose is present in an amount of about 50 to about 500 mg. In some embodiments, sucrose is present in an amount of about 200 to about 300 mg. In some embodiments, sucrose is present in an amount of about 250 mg. In some embodiments, sucrose is present in an amount of at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent by weight of the lyophilized composition. In some embodiments, sucrose is present in an amount of about 95% by weight of the lyophilized composition. In some embodiments, sucrose is present in an amount of 80-98%, 85-98%, 90-98%, or 94-96% by weight of the lyophilized composition.

[0113] In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is at a concentration of at least about 0.1% w / v. In some embodiments, the cryoprotectant is at a concentration of about 1% w / v to about 20% w / v. In some embodiments, the cryoprotectant is at a concentration of about 10% w / v to about 20% w / v. In some embodiments, the cryoprotectant is at a concentration of about 10% w / v.

[0114] In some embodiments, the compositions and vaccine compositions provided herein are thermally stable. A composition is considered to be thermally stable if the composition maintains its properties, such as the ability to resist the effects of heat or cold and protect nucleic acid molecules from degradation at a given temperature. In some embodiments, the compositions and vaccine compositions provided herein are thermally stable at about 25 degrees Celsius (°C) or standard room temperature. In some embodiments, the compositions and vaccine compositions provided herein are thermally stable at about 45°C. In some embodiments, the compositions and vaccine compositions provided herein are thermally stable at about -20°C. In some embodiments, the compositions and vaccine compositions provided herein are thermally stable at about 2°C to about 8°C. In some embodiments, the compositions and vaccine compositions provided herein are thermally stable at temperatures of at least about -80°C, at least about -20°C, at least about 0°C, at least about 2°C, at least about 4°C, at least about 6°C, at least about 8°C, at least about 10°C, at least about 20°C, at least about 25°C, at least about 30°C, at least about 37°C, and up to 45°C. In some embodiments, the compositions and vaccine compositions provided herein are thermally stable for at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 1 month, or up to 3 months. In some embodiments, the compositions and vaccine compositions provided herein are stored at a temperature of at least about 4° C. to a maximum of 37° C. for at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 1 month, or up to 3 months. In some embodiments, the compositions and vaccine compositions provided herein are stored at a temperature of at least about 20° C. to a maximum of 25° C. for at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 1 month, or up to 3 months.

[0115] Also provided herein is a method for preparing a lyophilized composition, the method comprising the steps of obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and lyophilizing the formulation to form a lyophilized composition.

[0116] Further provided herein is a method for preparing a spray-dried composition, the method comprising the steps of obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and spray drying the formulation to form the spray-dried composition.

[0117] Further provided herein is a method for reconstituting a lyophilized composition, the method comprising the steps of obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; lyophilizing the formulation to form a lyophilized composition; and reconstituting the lyophilized composition in a suitable diluent.

[0118] Further provided herein is a method for reconstituting a spray-dried composition, the method comprising the steps of obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; spray drying the formulation to form the spray-dried composition; and reconstituting the spray-dried composition in a suitable diluent. Pharmaceutical Compositions

[0119] Provided herein is a suspension comprising the composition provided herein. In some embodiments, the suspension provided herein comprises a number of nanoparticles or compositions provided herein. In some embodiments, the composition provided herein is a suspension, optionally a homogenous suspension. In some embodiments, the composition provided herein is in the form of an emulsion.

[0120] Also provided herein is a pharmaceutical composition comprising the composition provided herein.In some embodiments, the composition provided herein is combined with pharmaceutically acceptable salts, excipients, and / or carriers to form a pharmaceutical composition.Pharmaceutical salts, excipients, and carriers can be selected based on the route of administration, the location of target tissue, and the time course of drug delivery.Pharmaceutically acceptable carriers or excipients can include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.

[0121] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol). Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are used as solvents or dispersion media. For this purpose, any brand of fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0122] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or non-encapsulated conjugates contain at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dibasic calcium phosphate, and / or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; (c) humectants, such as glycerol; (d) agar, calcium carbonate, and / or (e) agar-agar, calcium carbonate, and / or (f) agar-agar, calcium carbonate, and / or (g) agar-agar, calcium carbonate, and / or (h) agar-agar, calcium carbonate, and / or (i) agar-agar, calcium carbonate, and / or (j) agar-agar, calcium carbonate, and / or (k) agar-agar, calcium carbonate, and / or (l ... The formulation may be mixed with disintegrating agents such as wheat flour, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) dissolution inhibitors such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents. dosage

[0123] The compositions provided herein can be formulated into unit dosage forms for ease of administration and uniformity of dosage.A unit dosage form is a physically separate unit of the compositions provided herein suitable for the subject to be treated.However, it will be understood that the overall use of the compositions provided herein is determined by the attending physician within the scope of good medical judgment.For any compositions provided herein, the therapeutically effective dose can be estimated first either in cell culture assays or in animal models such as mice, rabbits, dogs, pigs, or non-human primates.Animal models can also be used to achieve a desirable concentration range and route of administration.Such information can then be used to determine the useful dose and route for administration to humans.The therapeutic efficacy and toxicity of the compositions provided herein can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example, ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose that is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, or LD 50 / ED 50 The therapeutic index can be expressed as a ratio of 0.01 to 0.01. In some embodiments, pharmaceutical compositions that exhibit large therapeutic indices can be useful. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use. Administration

[0124] Provided herein are compositions and pharmaceutical compositions for administration to a subject in need thereof. In some embodiments, the pharmaceutical compositions provided herein are in a form that allows the compositions provided herein to be administered to a subject.

[0125] In some embodiments, the administration is local or systemic. In some embodiments, the compositions described herein are formulated for / use in administration via subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, intracranial, or intrathecal routes. In some embodiments, the administration is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours. In some embodiments, the administration is daily, weekly, or monthly. In some embodiments, the administration is repeated at least about every 28 or 56 days.

[0126] In some embodiments, a single dose of the composition provided herein is administered to the subject. In some embodiments, the composition or pharmaceutical composition provided herein is administered to the subject by two doses. In some embodiments, the second dose of the composition or pharmaceutical composition provided herein is administered about 28 days or 56 days after the first dose. In some embodiments, the first dose is administered and the second dose is administered about 14 days later, or about 21 days later, or about 28 days later, or about 35 days later, or about 42 days later, or about 49 days later, or about 56 days later, or about 63 days later, or about 70 days later, or about 77 days later, or about 84 days later. In some embodiments, the second dose is administered about 10-90 days after administration of the first dose, or about 15-85 days after administration of the first dose, or about 20-80 days after administration of the first dose, or about 25-75 days after administration of the first dose, or about 30-70 days after administration of the first dose, or about 35-65 days after administration of the first dose, or about 40-60 days after administration of the first dose.

[0127] In some embodiments, a third dose of a composition or pharmaceutical composition provided herein is administered to the subject. In some embodiments, the third dose is administered about 1 month after administration of the second dose, about 2 months after administration of the second dose, about 3 months after administration of the second dose, about 4 months after administration of the second dose, about 5 months after administration of the second dose, about 6 months after administration of the second dose, about 7 months after administration of the second dose, about 8 months after administration of the second dose, about 9 months after administration of the second dose, about 10 months after administration of the second dose, about 11 months after administration of the second dose, about 12 months after administration of the second dose, about 13 months after administration of the second dose, about 14 months after administration of the second dose, about 15 months after administration of the second dose, about 16 months after administration of the second dose, about 17 months after administration of the second dose, or about 18 months after administration of the second dose. Therapeutic Applications

[0128] Provided herein are methods for treating or preventing disease in a subject. In some embodiments, the compositions described herein are used to treat an infectious disease. In some embodiments, the compositions described herein are used to treat a respiratory infection. In some embodiments, the infectious disease is a viral infection. In some embodiments, the viral infection is from a coronavirus. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is MERS or SARS. In some embodiments, the coronavirus is a SARS-CoV-2 variant.

[0129] Further provided herein are methods for immunoprotecting a subject. In some embodiments, the methods reduce the severity of a SARS-CoV-2 infection. In some embodiments, the methods prevent SARS-CoV-2 infection in a subject. In some embodiments, the compositions described herein are used to enhance a subject's immune response to a viral antigen or variant thereof provided herein. In some embodiments, the compositions described herein are used to immunize a subject. In some embodiments, the compositions described herein are used to reduce the severity of an infection in a subject. In some embodiments, the compositions described herein result in a reduction in the severity or duration of symptoms associated with an infection in a subject.

[0130] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected of having a viral infection. In some embodiments, the subject has or is suspected of having a coronavirus infection. In some embodiments, the subject has or is suspected of having COVID-19 caused by the SARS-CoV-2 virus or a variant thereof. In some embodiments, the subject has COVID-19 caused by a delta variant of SARS-CoV-2. In some embodiments, the subject has COVID-19 caused by an omicron variant of SARS-CoV-2. In some embodiments, the subject is administered a composition provided herein comprising a nucleic acid encoding the stem helix region of the SARS-CoV-2 spike protein. In some embodiments, the subject is immunocompromised. In some embodiments, the subject is immunosuppressed (e.g., by an immunosuppressant) prior to administration. In some embodiments, the subject has received at least one dose of a SARS-CoV-2 vaccine prior to administration of a composition provided herein. In some embodiments, the subject has previously exhibited at least one symptom of an upper respiratory tract infection. kit

[0131] Provided herein is a kit comprising the composition provided herein, the pharmaceutical composition provided herein; and optionally a delivery system for administration to a subject. The kit described herein may comprise a lyophilized reagent, and optionally a reagent for hydration. The kit described herein may also comprise a non-lyophilized reagent. In some embodiments, the kit comprises two or more separate units, each comprising a lipid carrier and a nucleic acid. In some embodiments, the kit comprises a unit comprising a lipid carrier and a nucleic acid. In some embodiments, the kit further comprises a unit comprising a reagent for hydration of the dry composition. In some embodiments, the reagent for hydration comprises water.

[0132] In some embodiments, the kit further comprises one or more surfactants. In some embodiments, the formulation of the compositions described herein is prepared in a single container for administration. In some embodiments, the formulation of the compositions described herein is prepared in two containers for administration, separating the nucleic acid from the nanoparticle carrier. As used herein, "container" includes vessels, vials, ampoules, tubes, cups, boxes, bottles, flasks, jars, dishes, wells of single-well or multi-well instruments, reservoirs, tanks, etc., or other devices in which the compositions disclosed herein can be placed, stored, and / or transported, and accessed to remove contents. Examples of such containers include glass and / or plastic sealed or resealable tubes and ampoules, including those with rubber stoppers or other sealing means compatible with withdrawal of contents using a needle and syringe. In some implementations, the container is RNase-free.

[0133] In some embodiments, the kit includes: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more lipids, and one or more surfactants; and (b) at least one nucleic acid sequence comprising a sequence encoding a sequence capable of expressing an antigen, where the antigen is a SARS-CoV-2 spike protein. Exemplary embodiments

[0134] a lipid carrier comprising a liquid oil; and a surfactant, the surfactant comprising a cationic lipid; a hydrophilic surfactant; and a hydrophobic surfactant;As well as, compositions are provided herein comprising at least one nucleic acid, the at least one nucleic acid comprising a sequence at least 85% identical to SEQ ID NO: 1-7, optionally 1-8. Further provided herein are compositions wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO: 4, and optionally at least 85% identical to the sequence of SEQ ID NO: 27. Further provided herein are compositions wherein the nucleic acid comprises a sequence region that is SEQ ID NO: 4. Further provided herein are compositions wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO: 8. Further provided herein are compositions wherein the nucleic acid is identical to SEQ ID NO: 4. Further provided herein are compositions wherein the nucleic acid is identical to SEQ ID NO: 8. Further provided herein are compositions wherein the nucleic acid is complexed with a lipid carrier. Further provided herein are compositions wherein the nucleic acid further encodes an RNA polymerase. Further provided herein are compositions wherein the RNA polymerase is Venezuelan Equine Encephalitis Virus (VEEV) RNA polymerase. Further provided herein is a composition, wherein the nucleic acid encoding RNA polymerase comprises the nucleic acid sequence of SEQ ID NO: 20. Further provided herein is a composition, wherein the liquid oil is α-tocopherol, coconut oil, grape seed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E. Further provided herein is a composition, wherein the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, triglyceride ester, or triglycerol myristate. The cationic lipids were 1,2-dioleoyloxy-3(trimethylammonium)propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA);1,2-Dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearoyl trimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine ( DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 306Oi10, tetrakis(8-methylnonyl)3,3',3",3' ''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6 ,1-diyl)bis(2-hexyldecanoate);ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide;β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol;BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-cholesterol, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl) (oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide; cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate; DOPE, 1,2-di oleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl)9,9',9”,9'”,9''”,9''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl) Lipid H (SM-102), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9”Z,9''Z,12Z,12'Z,12”Z,12''Z)-tetrakis(octadeca-9,12-dienoate);Further provided herein is a composition which is PEG2000-DMG, (R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000) carbamate; TT3, or N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide;

[0135] Provided herein is a composition comprising a lipid carrier, the lipid carrier comprising a liquid oil; and a surfactant, the surfactant comprising a cationic lipid; a hydrophilic surfactant; and a hydrophobic surfactant; and at least one nucleic acid, the nucleic acid comprising a sequence encoding a SARS-CoV-2 omicron variant spike protein antigen or a functional variant thereof. Further provided herein is a composition, the SARS-CoV-2 omicron variant spike protein antigen or a functional variant thereof comprises a stem helix of a SARS-CoV-2 omicron variant spike protein antigen. Further provided herein is a composition, the SARS-CoV-2 omicron variant spike protein antigen stem helix comprises the amino acid sequence of SEQ ID NO: 19. Further provided herein is a composition, the nucleic acid encoding a SARS-CoV-2 omicron variant spike protein antigen comprises a polynucleotide sequence at least 85% identical to the sequence of SEQ ID NO: 8, and optionally at least 85% identical to the sequence of SEQ ID NO: 31. Further provided herein is a composition in which the SARS-CoV-2 omicron variant spike protein comprises the amino acid sequence of SEQ ID NO: 16. Further provided herein is a composition in which the nucleic acid is complexed with a lipid carrier. Further provided herein is a composition in which the nucleic acid further encodes an RNA polymerase. Further provided herein is a composition in which the RNA polymerase is Venezuelan Equine Encephalitis Virus (VEEV) RNA polymerase. Further provided herein is a composition in which the nucleic acid encoding the RNA polymerase comprises the nucleic acid sequence of SEQ ID NO: 20. Further provided herein is a composition in which the liquid oil is alpha-tocopherol, coconut oil, grape seed oil, lauroyl polyoxyl glyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E. Triglycerides include capric triglyceride, caprylic triglyceride,Further provided herein is a composition in which the cationic lipid is caprylic and capric triglycerides, triglyceride esters, or triglycerol myristate. The cationic lipid is selected from the group consisting of 1,2-dioleoyloxy-3(trimethylammonium)propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl (C16:0)trimethylammoniumpropane (DPTAP), distearoyltrimethylammoniumpropane (DS TAP), N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,1'-((2-(4 -(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 306Oi10, tetrakis(8-methylnonyl)3,3',3”,3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β-sitosterol,(3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol; BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-cholesterol, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide; cKK-E12, 3,6-bis(4-( Bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(spelt aminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate; Lipid H (SM-102),Heptadecane-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9”Z,9'''Z,12Z,12'Z,12”Z,12'' Further provided herein is a composition which is (Z)-tetrakis(octadeca-9,12-dienoate; PEG2000-DMG, (R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)carbamate; TT3, or N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide.

[0136] a lipid carrier comprising a liquid oil; and a surfactant, the surfactant comprising a cationic lipid; a hydrophilic surfactant; and a hydrophobic surfactant;As well as, provided herein is a composition comprising at least one nucleic acid, the nucleic acid comprising a sequence encoding a stem helix of a SARS-CoV-2 spike protein antigen or a functional variant thereof. Further provided herein is a composition, the SARS-CoV-2 spike protein antigen is derived from an alpha variant of SARS-CoV-2, a beta variant of SARS-CoV-2, a delta variant of SARS-CoV-2, a gamma variant of SARS-CoV-2, a mu variant of SARS-CoV-2, or an omicron variant of SARS-CoV-2. Further provided herein is a composition, the stem helix of the SARS-CoV-2 spike protein antigen comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19. Further provided herein is a composition, the nucleic acid is complexed with a lipid carrier. Further provided herein is a composition, the nucleic acid further encodes an RNA polymerase. Further provided herein is a composition, wherein the RNA polymerase is Venezuelan Equine Encephalitis Virus (VEEV) RNA polymerase. Further provided herein is a composition, wherein the nucleic acid encoding the RNA polymerase comprises the nucleic acid sequence of SEQ ID NO: 20. Further provided herein is a composition, wherein the liquid oil is α-tocopherol, coconut oil, grape seed oil, lauroyl polyoxyl glyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E. Further provided herein is a composition, wherein the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, triglyceride ester, or triglycerol myristate. The cationic lipids were 1,2-dioleoyloxy-3(trimethylammonium)propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA);1,2-Dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearoyl trimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine ( DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 306Oi10, tetrakis(8-methylnonyl)3,3',3",3' ''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6 ,1-diyl)bis(2-hexyldecanoate);ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide;β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol;BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-cholesterol, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl) (oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide; cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate; DOPE, 1,2-di oleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl)9,9',9”,9'”,9''”,9''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl) Lipid H (SM-102), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9”Z,9''Z,12Z,12'Z,12”Z,12''Z)-tetrakis(octadeca-9,12-dienoate);Further provided herein is a composition which is PEG2000-DMG, (R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000) carbamate; TT3, or N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide;

[0137] Provided herein is a composition comprising a lipid carrier, the lipid carrier comprising a liquid oil; inorganic nanoparticles, the inorganic nanoparticles comprising iron oxide present in an amount of about 0.2 milligrams per milliliter (mg / ml) of 12 nanometer (nm) iron oxide; and a surfactant, the surfactant comprising a cationic lipid; and at least one nucleic acid, the nucleic acid comprising a sequence encoding a SARS-CoV-2 spike protein antigen sequence or a functional variant thereof. The nucleic acid is present in an amount of up to about 200 micrograms (μg); the cationic lipid is present in a concentration of up to about 1.5 mg / ml; the iron oxide is present in a concentration of up to about 0.01 mg / ml; squalene is present in a concentration of up to about 1.88 mg / ml; sorbitan monostearate is present in a concentration of up to about 1.86 mg / ml; polysorbate 80 is present in a concentration of up to about 1.86 mg / ml;Further provided herein is a composition comprising sucrose present at a concentration of up to about 50mg / ml, and optionally citric acid monohydrate present at a concentration of up to about 2.1mg / ml.Further provided herein is a composition wherein the nucleic acid is RNA or DNA.Further provided herein is a composition wherein the nucleic acid is RNA and present at an amount of up to about 50μg.Further provided herein is a composition wherein the liquid oil is α-tocopherol, coconut oil, grape seed oil, lauroyl polyoxyl glyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E. Further provided herein is a composition, wherein the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, triglyceride ester, or triglycerol myristate.The cationic lipid is 1,2-dioleoyloxy-3(trimethylammonium)propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA);1,2-Dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearoyl trimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine ( DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 306Oi10, tetrakis(8-methylnonyl)3,3',3",3' ''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6 ,1-diyl)bis(2-hexyldecanoate);ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide;β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol;BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-cholesterol, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl) (oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide; cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate; DOPE, 1,2-di oleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl)9,9',9”,9'”,9''”,9''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl) Lipid H (SM-102), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9”Z,9''Z,12Z,12'Z,12”Z,12''Z)-tetrakis(octadeca-9,12-dienoate);Further provided herein is a composition in which the nucleic acid is complexed with a lipid carrier. Further provided herein is a composition in which the SARS-CoV-2 spike protein antigen is derived from an alpha variant of SARS-CoV-2, a beta variant of SARS-CoV-2, a delta variant of SARS-CoV-2, a gamma variant of SARS-CoV-2, a mu variant of SARS-CoV-2, or an omicron variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a variant of SARS-CoV-2 comprising the amino acid substitution D614G. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in one of SEQ ID NOs: 1-8. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein antigen sequence or a functional variant thereof has an amino acid sequence set forth in one of SEQ ID NOs: 9-16. Further provided herein is a composition, wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO: 4, and optionally at least 85% identical to the sequence of SEQ ID NO: 27. Further provided herein is a composition, wherein the nucleic acid further encodes an RNA polymerase. Further provided herein is a composition, wherein the RNA polymerase is Venezuelan Equine Encephalitis Virus (VEEV) RNA polymerase. Further provided herein is a composition, wherein the nucleic acid encoding the RNA polymerase comprises the nucleic acid sequence of SEQ ID NO: 20.;

[0138] Provided herein are compositions comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising about 30 mg / mL DOTAP chloride; about 37.5 mg / mL squalene; about 37 mg / ml sorbitan monostearate; about 37 mg / ml polysorbate 80; about 10 mM sodium citrate; and about 0.2 mg Fe / ml 12 nm oleic acid coated iron oxide nanoparticles; and (b) at least one nucleic acid, the at least one nucleic acid comprising a sequence encoding a SARS-CoV-2 spike protein antigen sequence or a functional variant thereof. Further provided herein is a composition comprising: a nucleic acid present in an amount of up to about 200 μg; a cationic lipid present in a concentration of up to about 1.5 mg / ml; an iron oxide present in a concentration of up to about 0.01 mg / ml; squalene present in a concentration of up to about 1.88 mg / ml; sorbitan monostearate present in a concentration of up to about 1.86 mg / ml; polysorbate 80 present in a concentration of up to about 1.86 mg / ml; sucrose present in a concentration of up to about 50 mg / ml; and optionally citric acid monohydrate present in a concentration of up to about 2.1 mg / ml. Further provided herein is a composition in which the nucleic acid is RNA or DNA. Further provided herein is a composition in which the nucleic acid is RNA and present in an amount of up to about 50 μg. Further provided herein is a composition further comprising sucrose. Further provided herein is a composition in which the sucrose is present in an amount of about 50 mg. Further provided herein is a composition, wherein the nucleic acid is complexed with a lipid carrier. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein antigen is derived from an alpha variant of SARS-CoV-2, a beta variant of SARS-CoV-2, a delta variant of SARS-CoV-2, a gamma variant of SARS-CoV-2, a mu variant of SARS-CoV-2, or an omicron variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a variant of SARS-CoV-2 that includes the amino acid substitution D614G.Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in one of SEQ ID NOs: 1-8. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein antigen sequence or a functional variant thereof has an amino acid sequence set forth in one of SEQ ID NOs: 9-17. Further provided herein is a composition, wherein the nucleic acid further encodes an RNA polymerase. Further provided herein is a composition, wherein the RNA polymerase is Venezuelan Equine Encephalitis Virus (VEEV) RNA polymerase. Further provided herein is a composition, wherein the nucleic acid encoding the RNA polymerase comprises a nucleic acid sequence set forth in SEQ ID NO: 20. Further provided herein is a composition, wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO: 4, and optionally at least 85% identical to the sequence of SEQ ID NO: 27.

[0139] Provided herein is a composition comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising: DOTAP chloride present in an amount of about 0.75 mg; squalene present in an amount of about 0.94 mg; sorbitan monostearate present in an amount of about 0.93 mg; polysorbate 80 present in an amount of about 0.93 mg; citric acid monohydrate present in an amount of about 1.05 mg; and oleic acid-coated iron oxide nanoparticles present in an amount of about 0.005 mg; and (b) at least one nucleic acid, the at least one nucleic acid comprising a sequence encoding a SARS-CoV-2 spike protein antigen sequence or a functional variant thereof. Provided herein further is a composition in which the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Provided herein further is a composition in which the at least one nucleic acid sequence is present in an amount of up to about 25 μg. Provided herein further is a composition further comprising sucrose. Further provided herein is a composition, wherein the sucrose is present in an amount of about 50 milligrams (mg). Further provided herein is a composition, wherein the nucleic acid is complexed with a lipid carrier. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein antigen is derived from an alpha variant of SARS-CoV-2, a beta variant of SARS-CoV-2, a delta variant of SARS-CoV-2, a gamma variant of SARS-CoV-2, a mu variant of SARS-CoV-2, or an omicron variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a variant of SARS-CoV-2 that includes an amino acid substitution of D614G. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in one of SEQ ID NOs: 1-8. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein antigen sequence or a functional variant thereof has an amino acid sequence set forth in one of SEQ ID NOs: 9-19. Further provided herein is a composition, wherein the nucleic acid further encodes an RNA polymerase.Further provided herein is a composition, wherein the RNA polymerase is Venezuelan Equine Encephalitis Virus (VEEV) RNA polymerase. Further provided herein is a composition, wherein the nucleic acid encoding the RNA polymerase comprises the nucleic acid sequence of SEQ ID NO:20. Further provided herein is a composition, wherein the composition is lyophilized. Further provided herein is a composition, wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO:4, and optionally at least 85% identical to the sequence of SEQ ID NO:27.

[0140] Provided herein is a suspension comprising a composition provided herein. Further provided herein is a pharmaceutical composition comprising a composition provided herein and a pharma- ceutically acceptable excipient.

[0141] Provided herein is a composition comprising: a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and b) at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Further provided herein is a composition wherein the nucleic acid is RNA. Further provided herein is a composition further comprising an additional nucleic acid encoding a nucleic acid polymerase or a sequence capable of expressing a nucleic acid polymerase. Further provided herein is a composition further comprising an RNA polymerase or a additional nucleic acid encoding a sequence capable of expressing an RNA polymerase. Further provided herein is a composition wherein the nucleic acid comprises a sequence set forth in one of SEQ ID NOs: 1-8. Further provided herein is a composition wherein the SARS-CoV-2 spike protein has an amino acid sequence set forth in one of SEQ ID NOs: 9-19. Further provided herein is a composition wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 1. Further provided herein is a composition wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 2. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO:3. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO:4. Further provided herein is a composition, wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO:4, and optionally at least 85% identical to the sequence of SEQ ID NO:27. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO:5. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO:6. Further provided herein is a composition, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO:7. Further provided herein is a composition, wherein the nucleic acid sequence comprises a sequence set forth in SEQ ID NO:8. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from an alpha variant of SARS-CoV-2.Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a beta variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a delta variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a gamma variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a mu variant of SARS-CoV-2. Further provided herein is a composition, wherein the SARS-CoV-2 spike protein is derived from a variant of SARS-CoV-2 that includes an amino acid substitution of D614G. Further provided herein is a composition, wherein the hydrophobic core comprises an oil. Further provided herein is a composition in which the oil comprises at least one of α-tocopherol, lauroyl polyoxyl glyceride, monoacyl glycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, and vitamin E, and medium chain triglyceride. Further provided herein is a composition in which the one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, metalloids, and any combination thereof. Further provided herein is a composition in which the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a composition in which the one or more lipids are cationic lipids.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N,N-dioleoyl-N, Further provided herein is a composition selected from the group consisting of N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C12-200), and any combination thereof. Further provided herein is a composition in which the lipid carrier optionally comprises one or more surfactants. Further provided herein is a composition in which the one or more surfactants are selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof. Further provided herein is a composition, wherein the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate, and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a composition, wherein the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm, and an average polydispersity index ranging from about 0.1 to about 0.4.Further provided herein is a composition in which one or more nucleic acids are incorporated into or complexed with a lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein is a composition in which the complex is formed by non-covalent or reversible covalent interactions. Further provided herein is a composition in which the molar ratio of lipid carrier to one or more nucleic acids, characterized by the nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:1. Further provided herein is a composition that is stable at 2-8 degrees Celsius.

[0142] Provided herein is a vaccine composition comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and (b) at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Further provided herein is a vaccine composition, the nucleic acid being RNA. Further provided herein is a vaccine composition, the nucleic acid being further comprising an additional nucleic acid encoding a nucleic acid polymerase or a sequence capable of expressing a nucleic acid polymerase. Further provided herein is a vaccine composition, the nucleic acid being further comprising an RNA polymerase or a sequence capable of expressing an RNA polymerase. Further provided herein is a vaccine composition, the nucleic acid being a sequence as set forth in one of SEQ ID NOs: 1-8. Further provided herein is a vaccine composition, the SARS-CoV-2 spike protein having an amino acid sequence as set forth in one of SEQ ID NOs: 9-17. Further provided herein is a composition, wherein the nucleic acid is at least 85% identical to the sequence of SEQ ID NO:4, and optionally at least 85% identical to the sequence of SEQ ID NO:27. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:1. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:2. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:3. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:4. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:5. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:6. Further provided herein is a vaccine composition, wherein the nucleic acid comprises the sequence set forth in SEQ ID NO:7. Further provided herein is a vaccine composition, wherein the nucleic acid sequence comprises the sequence set forth in SEQ ID NO:8.Further provided herein is a vaccine composition, wherein the SARS-CoV-2 spike protein is an alpha variant of SARS-CoV-2. Further provided herein is a vaccine composition, wherein the SARS-CoV-2 spike protein is a beta variant of SARS-CoV-2. Further provided herein is a vaccine composition, wherein the SARS-CoV-2 spike protein is a delta variant of SARS-CoV-2. Further provided herein is a vaccine composition, wherein the SARS-CoV-2 spike protein is a gamma variant of SARS-CoV-2. Further provided herein is a vaccine composition, wherein the SARS-CoV-2 spike protein is a mu variant of SARS-CoV-2. Further provided herein is a vaccine composition, wherein the SARS-CoV-2 spike protein is a D614G variant of SARS-CoV-2. Further provided herein is a vaccine composition, wherein the hydrophobic core comprises an oil. Further provided herein is a vaccine composition, wherein the oil comprises at least one of α-tocopherol, lauroyl polyoxyl glyceride, monoacyl glycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, vitamin E, medium chain triglyceride. Further provided herein is a vaccine composition, wherein the one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, metalloids, and any combination thereof. Further provided herein is a vaccine composition, wherein the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a vaccine composition, wherein the one or more lipids are cationic lipids.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP); dipalmitoyl (C16:0)trimethylammoniumpropane (DPTAP); distearoyltrimethylammoniumpropane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N- Further provided herein is a vaccine composition selected from the group consisting of dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C12-200), and any combination thereof. Further provided herein is a vaccine composition in which the lipid carrier optionally comprises one or more surfactants. Further provided herein is a vaccine composition in which the one or more surfactants are selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof. Further provided herein is a vaccine composition, wherein the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate, and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a vaccine composition, wherein the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm, and an average polydispersity index ranging from about 0.1 to about 0.4.Further provided herein is a vaccine composition in which one or more nucleic acids are incorporated into or complexed with a lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein is a vaccine composition in which the complex is formed by non-covalent or reversible covalent interactions. Further provided herein is a vaccine composition in which the molar ratio of lipid carrier to one or more nucleic acids, characterized by the nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:1. Further provided herein is a vaccine composition that is stable at 2-8 degrees Celsius.

[0143] Provided herein is a method of generating an immune response in a subject, comprising administering to the subject a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; b) a composition comprising at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Further provided herein is a method in which the composition is administered to the subject in two doses. Further provided herein is a method in which the second dose is administered about 28 days after the first dose. Further provided herein is a method in which the composition further comprises administering to the subject a third dose of the composition. Further provided herein is a method in which 5 micrograms of the composition are administered to the subject. Further provided herein is a method in which 10 micrograms of the composition are administered to the subject. Further provided herein is a method in which 25 micrograms of the composition are administered to the subject. Further provided herein is a method in which the subject is a mammal. Further provided herein is a method, wherein the mammal is a human.Further provided herein is a method, wherein the composition is administered intramuscularly.

[0144] Provided herein is a composition for immunoprotecting a subject, comprising: a) a lipid carrier, where the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and b) at least one nucleic acid sequence, where the nucleic acid sequence comprises a sequence capable of expressing an antigen, and where the antigen is a SARS-CoV-2 spike protein.

[0145] Provided herein is a method of reducing the severity of a SARS-CoV-2 infection, comprising administering a composition prior to infection comprising: a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and b) at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Further provided herein is a method in which the composition is administered to the subject in two doses. Further provided herein is a method in which the second dose is administered about 28 days after the first dose. Further provided herein is a method in which the composition further comprises administering a third dose of the composition to the subject. Further provided herein is a method in which 5 micrograms of the composition are administered to the subject. Further provided herein is a method in which 10 micrograms of the composition are administered to the subject. Further provided herein is a method in which 25 micrograms of the composition are administered to the subject. Further provided herein is a method in which the subject is a mammal. Further provided herein is a method, wherein the mammal is a human.Further provided herein is a method, wherein the composition is administered intramuscularly.

[0146] Provided herein is a method of immunoprotecting a subject, comprising: a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; and b) at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Further provided herein is a method, the composition is administered to the subject in two doses. Further provided herein is a method, the second dose is administered about 28 days after the first dose. Further provided herein is a method, the method further comprising administering a third dose of the composition to the subject. Further provided herein is a method, the composition is administered in 5 micrograms to the subject. Further provided herein is a method, the composition is administered in 10 micrograms to the subject. Further provided herein is a method, the composition is administered in 25 micrograms to the subject. Further provided herein is a method, the subject is a mammal. Further provided herein is a method, the mammal is a human. Further provided herein is a method, wherein the composition is administered intramuscularly.

[0147] Provided herein is a kit comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; (b) at least one nucleic acid sequence, the nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Provided herein is a kit comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more lipids, and one or more surfactants; (b) at least one nucleic acid sequence comprising a sequence encoding a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein. Provided herein is a kit further comprising one or more surfactants. Provided herein is a kit further comprising: an RNA comprising a sequence comprising a vector sequence as set forth in SEQ ID NO: 20. Provided herein is a kit further comprising: an RNA comprising a sequence comprising an antigen sequence as set forth in one of SEQ ID NOs: 1-8. Provided herein is a kit further comprising: an RNA comprising a sequence comprising a sequence as set forth in one of SEQ ID NOs: 24-31. Further provided herein is a kit, wherein the RNA comprises a sequence that includes a vector sequence set forth in one of SEQ ID NOs: 32-38.

[0148] Provided herein is a dry composition comprising: a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; b) at least one nucleic acid sequence comprising a sequence capable of expressing an antigen, the antigen being a SARS-CoV-2 spike protein; and c) at least one cryoprotectant. Further provided herein is a dry composition that is lyophilized. Further provided herein is a dry composition that is spray dried. Further provided herein is a dry composition that is thermally stable. Further provided herein is a dry composition that is thermally stable at about 25°C. Further provided herein is a dry composition that is thermally stable at about 45°C. Further provided herein is a dry composition that is thermally stable at about -20°C. Further provided herein is a dry composition that is thermally stable at about 2 degrees Celsius to about 8 degrees Celsius. Further provided herein is a dry composition that is thermally stable for at least one week, at least two weeks, and / or at least one month. Further provided herein is a dry composition, wherein the hydrophobic core comprises an oil. Further provided herein is a dry composition, wherein the oil comprises at least one of α-tocopherol, lauroyl polyoxyl glyceride, monoacyl glycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, vitamin E, medium chain triglyceride, dihydroisosqualene (DHIS), farnesene and squalane. Further provided herein is a dry composition, wherein the one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, metalloids, and any combination thereof. Further provided herein is a dry composition, wherein the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a dry composition, wherein the one or more lipids are cationic lipids.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium- Further provided herein is a dry composition selected from the group consisting of: propane (DODAP); and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC), and any combination thereof. Further provided herein is a dry composition, wherein the lipid carrier optionally comprises at least one surfactant. Further provided herein is a dry composition, wherein the at least one surfactant is selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof.Further provided herein is a dry composition, wherein the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of SPAN® 20, SPAN® 40, SPAN® 60, SPAN® 65, SPAN® 80 and SPAN® 85, and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a dry composition, wherein the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm, and an average polydispersity index ranging from about 0.1 to about 0.4. Further provided herein is a dry composition, wherein the one or more nucleic acid sequences are RNA. Further provided herein is a dry composition, wherein the RNA is a self-replicating RNA. Further provided herein is a dry composition, wherein the one or more nucleic acid sequences comprise a sequence encoding an antigen. Further provided herein is a dry composition, wherein the antigen is derived from a virus. Further provided herein is a dry composition, wherein the virus is selected from the group consisting of a hepatitis virus, a coronavirus, a mosquito-borne virus, and an HIV virus. Further provided herein is a dry composition in which one or more nucleic acid sequences are incorporated into or complexed with a lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein is a dry composition in which the lipid carrier-nucleic acid complex is formed by non-covalent or reversible covalent interactions. Further provided herein is a dry composition in which the molar ratio of lipid carrier to one or more nucleic acids, characterized by the nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:1. Further provided herein is a dry composition in which the at least one cryoprotectant is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof. Further provided herein is a dry composition in which the at least one cryoprotectant is sucrose. Further provided herein is a dry composition in which the at least one cryoprotectant is present at about 1% w / v to about 20% w / v.Further provided herein is a dry composition, wherein the at least one cryoprotectant is present at about 10% w / v to about 20% w / v. Further provided herein is a dry composition, wherein the at least one cryoprotectant is present at about 10% w / v.

[0149] Provided herein is a pharmaceutical composition comprising the composition provided herein reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier.Further provided herein is a pharmaceutical composition in which the diluent is aqueous.Further provided herein is a pharmaceutical composition in which the diluent is water.

[0150] Provided herein are kits that include the pharmaceutical compositions provided herein and a delivery system for administration to a subject.

[0151] Provided herein is a vaccine delivery system comprising a pharmaceutical composition provided herein and, optionally, one or more vaccine adjuvants.

[0152] Provided herein is a method for generating an immune response in a subject, comprising administering to the subject a composition or pharmaceutical composition provided herein, thereby generating an immune response to an antigen. Provided herein is a method in which the composition is administered to the subject in two doses. Provided herein is a method in which the second dose is administered about 28 days after the first dose. Provided herein is a method in which the subject further comprises administering to the subject a third dose of the composition. Provided herein is a method in which the subject is administered 5 μg of the composition. Provided herein is a method in which the subject is administered 10 μg of the composition. Provided herein is a method in which the subject is administered 25 μg of the composition. Provided herein is a method in which the composition is administered intramuscularly or intranasally. Provided herein is a method in which the subject is a human. Provided herein is a method in which the subject has or is suspected of having a viral infection. Provided herein is a method in which the viral infection is a coronavirus infection. Further provided herein is a method, wherein the coronavirus infection is caused by the SARS-CoV-2 virus or a variant thereof. Further provided herein is a method, wherein the antigen is the SARS-CoV-2 spike protein or a fragment thereof. Further provided herein is a method, wherein the immune response comprises an increase in the amount or titer of neutralizing antibodies against the antigen compared to a subject not receiving the composition. Further provided herein is a method, wherein the immune response comprises an increase in the amount of CD4+ and / or CD8+ positive T cells compared to a subject not receiving the composition. Further provided herein is a method, wherein the subject is immunocompromised or immunosuppressed.

[0153] Provided herein is a method of enhancing an immune response in a subject, the method comprising administering to the subject a composition provided herein, thereby enhancing the immune response to an antigen.

[0154] Provided herein is a method of treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0155] Provided herein is a method of imaging and / or tracking delivery of one or more nucleic acids in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0156] Provided herein is a method for preparing a lyophilized composition, the method comprising: a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; b) incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex, the nucleic acid comprising a sequence that comprises an antigen sequence at least 80% identical to one of SEQ ID NOs: 1-8, 24-31; c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and d) lyophilizing the formulation to form a lyophilized composition.

[0157] Provided herein is a method for preparing a spray-dried composition, the method comprising: (a) obtaining a lipid carrier, where the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; (b) incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex, where the nucleic acid comprises a sequence that comprises an antigen sequence that is at least 80% identical to one of SEQ ID NOs: 1-8, 24-31; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and (d) spray drying the formulation to form the spray-dried composition.

[0158] Provided herein is a method of reconstituting a lyophilized composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex, the nucleic acid comprising a sequence that comprises an antigen sequence at least 80% identical to one of SEQ ID NOs: 1-8, 24-31; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; (d) lyophilizing the formulation to form a lyophilized composition; and (e) reconstituting the lyophilized composition in a suitable diluent.

[0159] Provided herein is a method of reconstituting a spray-dried composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) incorporating at least one nucleic acid into the lipid carrier to form a lipid carrier-nucleic acid complex, the nucleic acid comprising a sequence comprising an antigen sequence at least 80% identical to one of SEQ ID NOs: 1-8, 24-31; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; (d) spray drying the formulation to form a spray-dried composition; and (e) reconstituting the spray-dried composition in a suitable diluent. Provided herein further is a method, the diluent is aqueous. Provided herein further is a method, the diluent is water. Provided herein further is a method, the lyophilized composition is thermally stable. Provided herein further is a method, the lyophilized composition is thermally stable at a temperature of up to about 25°C. Further provided herein is a method, wherein the freeze-dried composition is thermally stable at a temperature up to about 45°C. Further provided herein is a method, wherein the freeze-dried composition is thermally stable at a temperature ranging from about 2°C to about 8°C. Further provided herein is a method, wherein the freeze-dried composition is thermally stable for at least one week, at least two weeks, and / or at least one month. Further provided herein is a method, wherein the hydrophobic core comprises an oil. Further provided herein is a method, wherein the oil comprises at least one of α-tocopherol, lauroyl polyoxyl glyceride, monoacylglycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soybean lecithin, triglyceride, vitamin E, medium chain triglyceride, dihydroisosqualene (DHIS), farnesene, and squalane.Further provided herein is a method, wherein one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, metalloids, and any combination thereof.Further provided herein is a method, wherein one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof.Further provided herein is a method, wherein one or more lipids are cationic lipids. Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium -propane (DODAP); and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC); and any combination thereof.Further provided herein is a method in which the lipid carrier optionally comprises one or more surfactants. Further provided herein is a method in which the one or more surfactants are selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof. Further provided herein is a method in which the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of SPAN® 20, SPAN® 40, SPAN® 60, SPAN® 65, SPAN 80®, and SPAN® 85, and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a method in which the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm, and an average polydispersity index ranging from about 0.1 to about 0.4. Further provided herein is a method in which the one or more nucleic acids are RNA. Further provided herein is a method in which the RNA is a self-replicating RNA. Further provided herein is a method in which the one or more nucleic acids code for an antigen, the antigen being derived from a bacterial infection, a bacterial disease, a viral infection, a viral disease, a protozoan infection, a protozoan disease, a non-communicable disease, one or more cancers, or an autoimmune disease. Further provided herein is a method in which the antigen is derived from a virus. Further provided herein is a method in which the virus is selected from the group consisting of a hepatitis virus, a coronavirus, a mosquito-borne virus, and an HIV virus. Further provided herein is a method in which the one or more nucleic acids are incorporated into or complexed with a lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein is a method in which the lipid carrier-nucleic acid complex is formed by a non-covalent or reversible covalent interaction. Further provided herein is a method in which at least one cryoprotectant is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof. Further provided herein is a method in which at least one cryoprotectant is sucrose.Further provided herein is a method, wherein the at least one cryoprotectant is present at about 1% w / v to about 20% w / v. Further provided herein is a method, wherein the at least one cryoprotectant is present at about 10% w / v to about 20% w / v. Further provided herein is a method, wherein the at least one cryoprotectant is present at about 10% w / v.

[0160] Provided herein is a composition for the prevention of SARS-CoV-2, comprising: a) a sorbitan fatty acid ester; b) an ethoxylated sorbitan ester; c) a cationic lipid; d) an immunostimulant; and e) at least one RNA encoding an antigen sequence or a functional fragment thereof. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the sorbitan fatty acid ester is sorbitan monostearate. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the ethoxylated sorbitan ester is TWEEN® 80. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the cationic lipid is DOTAP. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the immunostimulant is squalene. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the RNA encodes the SARS-CoV-2 spike protein. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the ratio of esters results in a hydrophilic lipophilic balance between 8 and 11. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the ratio of esters to lipids results in a particle size between 30 nanometers and 200 nanometers. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the ratio of esters to lipids results in a particle size between 40 nanometers and 70 nanometers. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the immunostimulant reduces the total amount of protein produced, but increases the immune response to the vaccine. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the immunostimulant increases the total amount of protein produced, but decreases the immune response to the vaccine. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the immunostimulant is Miglyol 810 or Miglyol 812.Further provided herein is a composition for the prevention of SARS-CoV-2, further comprising sorbitan monostearate, polysorbate 80, DOTAP, and squalene, and not comprising solid particles.

[0161] Provided herein is a composition for the prevention of SARS-CoV-2, comprising: (a) sorbitan monostearate; (b) polysorbate 80; (c) DOTAP; (d) an immunostimulant; and (e) at least one RNA encoding an antigen sequence or a functional fragment thereof. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the immunostimulant reduces the total amount of protein produced, but increases the immune response to the vaccine. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the immunostimulant increases the total amount of protein produced, but decreases the immune response to the vaccine. Further provided herein is a composition for the prevention of SARS-CoV-2, in which the immunostimulant is Miglyol 810 or Miglyol 812. Further provided herein is a composition for the prevention of SARS-CoV-2, further comprising squalene and not comprising solid particles. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the ratio of esters results in a hydrophilic lipophilic balance between 8 and 11. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the particle size is between 30 nanometers and 200 nanometers. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the N to P ratio is between 5 and 35. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the RNA encodes an amino acid sequence set forth in one of SEQ ID NOs: 9-19. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the RNA comprises a sequence set forth in one of SEQ ID NOs: 1-8. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the RNA comprises a sequence set forth in one of SEQ ID NOs: 24-31. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the RNA comprises a sequence comprising a vector sequence set forth in SEQ ID NO: 20.Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the RNA comprises a sequence comprising a sequence set forth in one of SEQ ID NOs: 1-8, 24-31. Further provided herein is a composition for the prevention of SARS-CoV-2, wherein the RNA comprises a sequence comprising a vector sequence set forth in one of SEQ ID NOs: 32-38. Further provided herein is a composition for the prevention of SARS-CoV-2, comprising a nucleic acid comprising an alphavirus replicon.

[0162] Provided herein is a dry composition comprising a lipid carrier, which is a nanoemulsion comprising a hydrophobic core and one or more lipids; and at least one nucleic acid comprising a sequence capable of expressing an antigen, where optionally the antigen is a SARS-CoV-2 spike protein or a functional variant thereof; and (i) at least one sugar present in an amount of at least about 50% by weight of the dry composition, or (ii) in an amount of at least 50 mg. Further provided herein is a composition that is lyophilized. Further provided herein is a composition that is thermally stable at about 25 degrees Celsius. Further provided herein is a composition that is thermally stable at about 45 degrees Celsius. Further provided herein is a composition that is thermally stable at about -20 degrees Celsius. Further provided herein is a composition that is thermally stable at a temperature between about 2 degrees Celsius and about 8 degrees Celsius. Further provided herein is a composition that is thermally stable for at least one week, at least two weeks, and / or at least one month. Further provided herein is a composition in which the hydrophobic core comprises an oil. Further provided herein is a composition in which the oil comprises at least one of α-tocopherol, lauroyl polyoxyl glyceride, monoacyl glycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, vitamin E, caprylic / capric triglyceride, triglyceride esters of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and plant-derived glycerol, dihydroisosqualene (DHIS), farnesene, and squalane. Further provided herein is a composition in which the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a composition in which the one or more lipids comprise cationic lipids.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium Further provided herein is a composition selected from the group consisting of N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC); and any combination thereof. Further provided herein is a composition, wherein the lipid carrier comprises at least one surfactant. Further provided herein is a composition, wherein the at least one surfactant is selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof.Further provided herein is a composition in which the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate, and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a composition in which the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm, and an average polydispersity index ranging from about 0.1 to about 0.4. Further provided herein is a composition in which the one or more nucleic acids are DNA. Further provided herein is a composition in which the one or more nucleic acids are RNA. Further provided herein is a composition in which the RNA is a self-replicating RNA. Further provided herein is a composition in which the hydrophobic core comprises one or more inorganic nanoparticles. Further provided herein is a composition in which the one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, and any combination thereof. Further provided herein is a composition in which one or more nucleic acids are incorporated into or complexed with lipid carrier to form lipid carrier-nucleic acid complex. Further provided herein is a composition in which lipid carrier-nucleic acid complex is formed by non-covalent or reversible covalent interaction. Further provided herein is a composition in which the molar ratio of lipid carrier to one or more nucleic acids, characterized by nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:1. Further provided herein is a composition in which at least one sugar is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof.Further provided herein is a composition in which at least one sugar is present in an amount of at least about 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, 300mg or more. Further provided herein is a composition in which at least one sugar is present in an amount of 50mg to 250mg. Further provided herein is a composition in which at least one sugar is present in an amount of at least about 250mg. Further provided herein is a composition, wherein the sugar is present in an amount of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more by weight of the composition. Further provided herein is a composition, wherein the sugar is present in an amount of 80-98%, optionally 94-96% by weight of the composition. Further provided herein is a composition, wherein the sugar is present in an amount of about 95% by weight of the composition. Further provided herein is a composition, wherein at least one sugar comprises sucrose. Further provided herein is a pharmaceutical composition, comprising a dry composition disclosed herein reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier. Further provided herein is a pharmaceutical composition, wherein the diluent is aqueous. Further provided herein is a pharmaceutical composition, wherein the diluent is water. Further provided herein is a kit, comprising a pharmaceutical composition as described herein and a delivery system for administration to a subject. Further provided is a method for generating an immune response in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition described herein.Further provided is a method for treating or preventing a disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition described herein.Further provided herein is a method of imaging and / or tracking the delivery of one or more nucleic acids in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition described herein. Further provided herein is a composition comprising a first nucleic acid comprising a sequence encoding an RNA-dependent RNA polymerase and a second nucleic acid sequence encoding a SARS-CoV-2 omicron antigen sequence. Further provided herein is a composition in which the first nucleic acid sequence and the second nucleic acid sequence are present on a shared nucleic acid. Further provided herein is a composition in which the first nucleic acid sequence and the second nucleic acid sequence are present on separate nucleic acids. Further provided herein is a composition in which the RNA-dependent RNA polymerase comprises VEEV RNA polymerase. Further provided herein is a composition further comprising a nanoparticle carrier system. Further provided herein is a composition in which the nanoparticle carrier system comprises a cationic lipid and a hydrophobic core. Further provided herein is a composition in which the hydrophobic core comprises an inorganic nanoparticle. Further provided herein is a composition in which the first nucleic acid and / or the second nucleic acid comprises RNA. Further provided herein is a composition, wherein the second nucleic acid sequence comprises a sequence at least 85% identical to SEQ ID NO:8. Further provided herein is a composition, comprising a first nucleic acid comprising a sequence encoding an RNA-dependent RNA polymerase, and a second nucleic acid sequence encoding a SARS-CoV-2 spike S2 ​​sequence region, optionally a stem-helix region. Further provided herein is a composition, wherein the first nucleic acid and the second nucleic acid are present on a shared nucleic acid. Further provided herein is a composition, wherein the first nucleic acid and the second nucleic acid sequence are present on separate nucleic acids. Further provided herein is a composition, wherein the RNA-dependent RNA polymerase comprises VEEV RNA polymerase. Further provided herein is a composition, further comprising a nanoparticle carrier system. Further provided herein is a composition, wherein the nanoparticle carrier system comprises a cationic lipid and a hydrophobic core. Further provided herein is a composition, wherein the hydrophobic core comprises an inorganic nanoparticle.Further provided herein is a composition, wherein the first nucleic acid and / or the second nucleic acid comprises RNA. Further provided herein is a composition, wherein the second nucleic acid sequence comprises a sequence at least 85% identical to SEQ ID NO: 18. Further provided herein is a composition, wherein the second nucleic acid sequence comprises a sequence at least 85% identical to SEQ ID NO: 19. Further provided herein is a method of treating or preventing a disease in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0163] The following examples are set forth to more clearly illustrate to one skilled in the art the principles and practice of the embodiments disclosed herein, and are not to be construed as limiting the scope of any claimed embodiments. All parts and percentages are by weight unless otherwise specified. EXAMPLES

[0164] Example 1 Macrophage-mediated immune response Various formulations containing lipid carriers and repRNA were prepared and analyzed to evaluate the innate immune response to the lipid carriers by macrophages. Protein expression and stimulation of tumor necrosis factor alpha (e.g., TNF-α) production in THP-1 macrophages were assayed.

[0165] First, THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA). Then, cells were transfected with various formulations with replicon RNA encoding NanoLuciferase (NanoLuc). Cell culture media was then evaluated for NanoLuc and TNFα expression.

[0166] The formulations used in this assay and their characteristics, such as particle size and PDI, are listed in Table 4. The concentration of repRNA encoding NanoLuc was 909 nanograms per microliter (ng / μl) and maintained at -80 degrees Celsius. Miglyol 812N (caprylic / capric triglyceride) was used in this example. [Table 4]

[0167] Fe-lipid carrier formulation-NP-1 (prepared at 100 ml scale): The Fe-lipid carrier formulation contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml 12 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems, San Diego, CA, USA) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 12 nm diameter oleic acid coated iron oxide nanoparticles in 1 ml chloroform (Imagion Biosystems, lot number 95-127) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase.The mixture was emulsified using a VWR® 200 homogenizer (VWR International, Radnor, PA, USA) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics, Westwood, MA, USA) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.25. The microfluidized NP-1 formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES). DOTAP and squalene concentrations were measured by reversed-phase high performance liquid chromatography (RP-HPLC).

[0168] High Fe-lipid carrier formulation NP-2 (prepared at 100 ml scale): The high Fe-lipid carrier formulation contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 1 mg Fe / ml 15 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles in 5 ml chloroform (Imagion Biosystems, lot number 95-133) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase.The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP and squalene concentrations were measured by RP-HPLC. Example 2 Fe-lipid carrier miglyol formulation NP-3 (prepared on a 100ml scale)

[0169] The Fe-lipid carrier miglyol formulation contained 37.5 mg / ml Miglyol 812 N (IOI Oleo GmbH), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml 15 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles in 1 ml chloroform (Imagion Biosystems, lot number 95-127) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting coarse emulsion was processed by passing it through an M110P Microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and a secondary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, as measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3.The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP concentrations were measured by RP-HPLC. Example 3 High Fe-lipid carrier Miglyol formulation NP-4 (prepared at 100ml scale)

[0170] The high Fe-lipid carrier Miglyol formulation contains 37.5 mg / ml Miglyol 812 N (IOI Oleo GmbH), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 1 mg / ml 15 nm oleic acid coated iron oxide nanoparticles (ImagionBio) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles in 5 ml chloroform (ImagionBio, lot number 95-127) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting coarse emulsion was processed by passing it through an M110P Microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and a secondary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, as measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3.The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP concentrations were measured by RP-HPLC. Example 4 Alum-lipid carrier formulation NP-5 (prepared at 100ml scale)

[0171] The alum-lipid carrier formulation contained 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 1 mg Al / ml TOPO-coated Alhydrogel® (aluminum oxyhydroxide) particles (Croda) and 10 mM sodium citrate. 10 ml of Alhydrogel was washed three times in methanol by centrifugation at 1000 rpm for 20 minutes. After the third wash, the Alhydrogel was dispersed in 10 ml of methanol, and 1 ml of 250 mg / ml trioctylphosphine oxide (TOPO) was added to the dispersion and incubated overnight at 37° C. on an orbital shaker. Excess TOPO was removed by an additional methanol wash and then dispersed in 11 ml of methanol. The methanol was allowed to evaporate overnight in a fume hood leaving a dry layer of TOPO-Alhydrogel. To this dry TOPO-Alhydrogel layer, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase.The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Aluminum concentrations were determined by ICP-OES. DOTAP and squalene concentrations were measured by RP-HPLC. Example 5 Fe-lipid carrier Solanesol formulation NP-6 (prepared at 100ml scale)

[0172] The Fe-lipid carrier solanesol formulation (NP-6) contains 37.5 mg / ml solanesol (Cayman chemicals), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml oleic acid coated iron oxide nanoparticles (ImagionBio) and 10 mM sodium citrate. 20 mg Fe / ml oleic acid coated iron oxide nanoparticles with a diameter of 15 nm in 1 ml chloroform (ImagionBio, lot number 95-133) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood leaving a coating of dry oleic acid iron oxide nanoparticles. To this iron oxide coating, 3.75 grams of Solanesol, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65 degree Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through a M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi. The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP and solanesol concentrations were measured by RP-HPLC. Example 6 NP-7 formulation (prepared at 100ml scale)

[0173] The NP-7 formulation contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 2.4 mg / ml Dynasan 114 (IOI Oleo GmbH) and 10 mM sodium citrate. To a 200 ml beaker, 3.75 grams of squalene, 3.7 grams of SPAN® 60, 0.24 grams of Dynasan 114 and 3 grams of DOTAP were added to generate an oil phase. The oil phase was sonicated in a 65 degree Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. DOTAP and squalene concentrations were measured by RP-HPLC. Example 7 NP-8 formulation (prepared at 100ml scale)

[0174] The NP-8 formulation contains 43 mg / ml squalene (SEPPIC), 5 mg / ml SPAN® 85 (Millipore Sigma), 5 mg / ml TWEEN® 80 (Fisher Chemical), 4 mg / ml DOTAP chloride (LIPOID), and 10 mM sodium citrate. In a 200 ml beaker, 4.3 grams of squalene, 0.5 grams of SPAN® 85, and 0.4 grams of DOTAP were added to generate an oil phase. The oil phase was sonicated in a 65 degrees Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 2.6 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 95 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 95 ml of the water phase by adding the warm oil phase to the warm water phase. The mixture was emulsified using a VWR® 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through a M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and a supplementary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter reached 100 ± 10 nm with a polydispersity index (PDI) of 0.05-0.1 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. DOTAP and squalene concentrations were measured by RP-HPLC.

[0175] Treatment groups were prepared. Eight of these groups were NanoLuc repRNA groups, with 600ng dose per well prepared using Fe-lipid carrier, high Fe-lipid carrier, Fe-lipid carrier miglyol, high Fe-lipid carrier miglyol, alum-lipid carrier, Fe-lipid carrier solanesol (SLN), NLC, and CNE formulations. The untreated group did not have NanoLuc.

[0176] Various formulations were prepared by diluting NanoLuc repRNA to 8 ng / μL in 2.2 mL of RNAse-free water. The lipid carrier formulation and RNA master mix were complexed by adding 250 μL of each diluted formulation and 250 μL of diluted RNA and mixed by pipetting up and down.

[0177] Cell transfections were performed at 7 × 10 cells per well in 24-well plates. 5 The transfection was performed by seeding 100 THP-1s. 80 micromolar (μM) PMA was added to each well and incubated at 37 degrees Celsius. The following day, 1 hour before transfection, the PMA-containing medium was removed and replaced with complete RPMI (cRPMI) medium. Samples were then serially diluted in Opti-MEM™ (Thermo Fisher Scientific, Waltham, MA USA) to generate a 10-point 1.5-fold dilution series starting at 0.45 ng / μL. Culture medium was then removed from the plate by pipetting. 450 μL of Opti-MEM™ and 150 μL of complexed formulation were added to the plate in duplicate. Empty wells received 450 μL of Opti-MEM™ only. After 4 hours, samples were removed from the plate by pipetting and replaced with 500 μL of growth medium. The plates were then incubated overnight at 37 degrees C. Growth medium was collected the next day and stored at -80 degrees C. Downstream assays were performed and described below.

[0178] Luciferase assays were performed by first diluting Nano-Glo® Luciferase Assay Reagent 1:50 in buffer. 25 μL of supernatant was removed and mixed with 25 μL of Nano-Glo® Reagent in a 96-well plate. This was incubated at room temperature for 3 minutes. Luminescence was read using a luminometer.

[0179] An ELISA assay was then performed to assess TNF-alpha (α) protein levels in cell culture media using Human TNF-α DUOSET™ ELISA (R&D Systems) according to the manufacturer's protocol. A 96-well microplate was coated with anti-TNFα capture antibody. The plate was blocked, and then medium samples were added directly without dilution. After addition of biotinylated detection antibody SA-HRP and substrate, absorbance was read at 450 nm in a SPECTRAMAX® i3 (Molecular Devices) plate reader.

[0180] In this example, all tests were performed in duplicate. The results from the duplicate tests are presented as the first and second experiments, respectively. As shown in the first experiment in FIG. 4A and the second experiment in FIG. 4B, the formulation containing lipid carrier and miglyol induced high protein production from the replicon. A reduction in the innate immune response, measured by TNF-α secretion, was detected (FIGS. 5A-5B).

[0181] As shown in the first experiment in Figure 6A and the second experiment in Figure 6B, a correlation between enhanced protein production and reduced TNF-alpha stimulation was observed when using the miglyol lipid carrier formulation. Protein production induced by the solanesol lipid carrier formulation was slightly lower, whereas TNFα production was high (Figures 6A-6B). The correlation data from the first assay shown in Figure 6A was derived from the data shown in Figures 4A and 5A. The correlation data from the second assay is shown in Figure 6B, and this correlation data was derived from the data shown in Figures 4B and 5B. Example 8 Techniques and Materials for Making Lipid Carriers

[0182] The following materials were used in the preparation of lipid-inorganic nanoparticles (i.e., lipid carriers): The compositions, kits and methods described herein are not limited to the techniques or materials described herein.

[0183] Iron oxide nanoparticles with various average diameters (5 nm, 10 nm, 15 nm, 20 nm, 25 nm and 30 nm) at 25 mg / ml Fe in chloroform were purchased from Ocean Nanotech (San Diego, CA, USA). Squalene and SPAN® 60 (sorbitan monostearate) were purchased from Millipore Sigma. TWEEN® 80 (polyethylene glycol sorbitan monooleate) and sodium citrate dihydrate were purchased from Fisher Chemical. The chloride salt of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP chloride) was purchased from Corden Pharma. Ultrapure water (18.2 megaohm-centimeter (MOhm-cm) resistivity) was obtained from a Milli-Q water purification system (Millipore Sigma).

[0184] Lipid carriers include squalene, sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP chloride, iron oxide nanoparticles, and sodium citrate dihydrate. Typically, chloroform was added to iron oxide nanoparticles with a number-weighted average diameter of 5 nm. The chloroform was allowed to evaporate in a fume hood, leaving a dry coating of iron oxide nanoparticles. SPAN® 60, squalene, and DOTAP chloride were added to the iron oxide nanoparticles to prepare the "oil" phase.

[0185] The oil phase was sonicated for 30 minutes in a water bath preheated to 60° C. Separately, a "water" phase was prepared in a 1 liter glass bottle by adding TWEEN® 80 to a sodium citrate dihydrate solution prepared with Milli-Q water.

[0186] The aqueous phase was stirred for 30 minutes to completely dissolve the TWEEN® 80. Once the TWEEN® 80 was completely dissolved, the aqueous phase was transferred to a beaker and incubated in a preheated water bath at 60° C. The preheated aqueous phase was added to the heated oil phase.

[0187] The mixture was immediately emulsified using a VWR® 200 homogenizer (VWR International) until a uniform colloid with a milky appearance was produced. The colloid was then processed by passing the fluid through the Y-shaped interaction chamber of an LM10 Microfluidizer at 20,000 psi.

[0188] The fluid was passed until the z-average hydrodynamic diameter, as measured by dynamic light scattering (Malvern Zetasizer Nano S), was 59 nm with a polydispersity index of 0.2.

[0189] The microfluidized lipid carrier samples were finally filtered using a polyethersulfone (PES) syringe filter with a pore size of 200 nm. Example 9 Exemplary Techniques and Materials for Making Lipid Carriers

[0190] The following materials were used in the preparation of lipid-inorganic nanoparticles (i.e., lipid carriers): The compositions, kits and methods described herein are not limited to the techniques or materials described herein.

[0191] Iron oxide nanoparticles with various average diameters (5 nm, 10 nm, 15 nm, 20 nm, 25 nm and 30 nm) at 25 mg / ml Fe in chloroform were purchased from Ocean Nanotech (San Diego, CA). Squalene and SPAN® 60 (sorbitan monostearate) were purchased from Millipore Sigma. TWEEN® 80 (polyethylene glycol sorbitan monooleate) and sodium citrate dihydrate were purchased from Fisher Chemical. The chloride salt of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP chloride) was purchased from Corden Pharma. Ultrapure water (resistivity of 18.2 MOhm-cm) was obtained from a Milli-Q water purification system (Millipore Sigma).

[0192] A lipid carrier was prepared containing 37.5 mg / ml squalene, 37 mg / ml SPAN® 60, 37 mg / ml TWEEN® 80, 30 mg / ml DOTAP chloride, 0.1 mg / ml 10 nm iron oxide nanoparticles and 10 mM sodium citrate dihydrate.

[0193] The lipid carrier was prepared using the following procedure: In a 200 ml beaker, 0.4 ml of iron oxide nanoparticles of 10 nm number-weighted average diameter, 25 mg Fe / ml in chloroform, was added.

[0194] The chloroform was allowed to evaporate in a fume hood leaving a dry coating of iron oxide nanoparticles. An "oil" phase was prepared by adding 3.7 grams of SPAN® 60, 3.75 grams of squalene, and 3 grams of DOTAP chloride to the iron oxide nanoparticles.

[0195] The oil phase was sonicated for 30 minutes in a water bath preheated to 60° C. Separately, in a 1 liter glass bottle, a "water" phase was prepared by adding 39 grams of TWEEN® 80 to 1,000 ml of a 10 mM sodium citrate dihydrate solution prepared with Milli-Q water.

[0196] The aqueous phase was stirred for 30 minutes to allow the TWEEN® 80 to completely dissolve. Once the TWEEN® 80 was completely dissolved, 96 ml of the aqueous phase was transferred to a 200 ml beaker and incubated in a water bath preheated to 60° C. To the heated oil phase, 96 ml of the preheated aqueous phase was added. The mixture was immediately emulsified using a VWR® 200 homogenizer (VWR International) until a uniform colloid with a milky appearance was produced. The colloid was then processed by passing the fluid through the Y-shaped interaction chamber of an LM10 microfluidizer at 20,000 psi. The fluid was passed until the z-average hydrodynamic diameter was 54 nm with a polydispersity index of 0.2 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized lipid carrier sample was finally filtered using a 200 nm pore size polyethersulfone (PES) syringe filter. Example 10 Exemplary Techniques and Materials for Making the Nanoparticles Described Herein

[0197] A lipid carrier was prepared containing 37.5 mg / ml squalene, 37 mg / ml SPAN® 60, 37 mg / ml TWEEN® 80, 30 mg / ml DOTAP chloride, 0.2 mg / ml 15 nm iron oxide nanoparticles, and 10 M sodium citrate dihydrate. The lipid carrier of Example 9 was manufactured using the following procedure.

[0198] In a 200 ml beaker, 0.8 ml of iron oxide nanoparticles with a number-weighted average diameter of 15 nm, 25 mg Fe / ml in chloroform, was added. The chloroform was allowed to evaporate in a fume hood, leaving a dry coating of iron oxide nanoparticles. To the iron oxide nanoparticles, 3.7 grams of SPAN® 60, 3.75 grams of squalene, and 3 grams of DOTAP chloride were added to prepare the "oil" phase.

[0199] The oil phase was sonicated for 30 minutes in a water bath preheated to 60° C. Separately, a “water” phase was prepared in a 1 liter glass bottle by adding 39 grams of TWEEN® 80 to 1,000 ml of 10 mM sodium citrate dihydrate solution prepared with Milli-Q water. The water phase was stirred for 30 minutes to completely dissolve the TWEEN® 80.

[0200] Once the TWEEN® 80 was completely dissolved, 96 ml of the aqueous phase was transferred to a 200 ml beaker and incubated in a preheated water bath at 60° C. To the heated oil phase, 96 ml of the preheated aqueous phase was added. The mixture was immediately emulsified using a VWR® 200 homogenizer (VWR International) until a uniform colloid with a milky appearance was produced. The colloid was subsequently processed by passing the fluid through the Y-shaped interaction chamber of an LM10 microfluidizer at 20,000 psi.

[0201] Fluid was passed until the z-average hydrodynamic diameter, as measured by dynamic light scattering (Malvern Zetasizer Nano S), was 52 nm with a polydispersity index of 0.2. The microfluidized lipid carrier samples were final filtered using a 200 nm pore size polyethersulfone (PES) syringe filter. Example 11 Lipid carrier formulation without inorganic core (prepared at 100 mL scale)

[0202] Described herein is a lipid carrier formulation without inorganic core particles that contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID) and 10 mM sodium citrate.

[0203] The composition was prepared as follows: 3.75 grams of squalene, 3.7 grams of SPAN 60®, and 3.0 grams of DOTAP were added to a 200 ml beaker to form an oil phase. The oil phase was sonicated in a 65° C. water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 96 ml of the aqueous phase was transferred to another glass bottle and heated at 65 degrees Celsius for 30 minutes. The oil phase and 96 ml of the aqueous phase were mixed by adding the warm oil phase to the warm aqueous phase.

[0204] The mixture was then emulsified using a VWR® 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through a M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter measured by dynamic light scattering (Malvern Zetasizer Nano S) reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3. The microfluidized lipid carrier composition (without inorganic core) formulation was then final filtered using a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius (°C). DOTAP and squalene concentrations were then measured by RP-HPLC. Example 12 Exemplary Techniques and Materials for Making the Nanoparticles Described Herein

[0205] In the mouse assay, C57BL / 6 mice were inoculated as described in Table 6 below, and then serum secreted embryonic alkaline phosphatase (SEAP) levels were measured. A summary of the materials used in this example is provided in Table 5. [Table 5] [Table 6]

[0206] Seven different formulations were prepared and administered intramuscularly across seven treatment groups (Groups 1-7). DNA-SEAP or RNA-SEAP was diluted according to the volumes listed in Table 7 to prepare the formulations for Groups 1-7. [Table 7]

[0207] The concentrations of the diluted DNA or RNA before complexing with lipid carriers (measured by NanoDrop spectrophotometer) were as follows: Groups 1, 4 and 5 contained approximately 820 μg / ml of DNA, Groups 2 and 3 contained approximately 480 μg / ml of DNA, and Groups 6 and 7 contained approximately 43 μg / ml of RNA.

[0208] Formulations for Groups 1-7 were diluted with 100 mM citric acid as listed in Table 8 below. [Table 8]

[0209] The above formulations were complexed by adding 250 μl of diluted lipid carrier to 250 μl of diluted DNA or RNA. The resulting complexed formulations were incubated on ice for at least 30 minutes. Table 9 describes the experimental schedule for the assay. [Table 9]

[0210] Mice were bled at regular intervals and serum was immediately prepared and stored at -80 degrees Celsius until analysis for SEAP activity.

[0211] To evaluate the SEAP levels in serum, all serum samples were thawed at the same time and SEAP detection was performed. Figures 7A-7F illustrate the SEAP levels in BALB / c mice intramuscularly injected with DNA SEAP formulated with various replicates of lipid carriers. Mice were bled at regular intervals, and serum was prepared and stored until analysis by SEAP assay. Data are presented as mean and SE (n=5 per group).

[0212] As can be seen from Figures 7A-7F, the lipid carrier formulation supported target protein production compared to delivery of DNA alone, especially after day 6 post-injection. Furthermore, the data show that incorporation of miglyol enhances protein production from RNA replicons compared to lipid carrier formulations lacking miglyol. Example 13 Additional nanoparticle formulations

[0213] Additional nanoparticle formulations are made according to the following tables (Tables 10 and 11). In a first example, the mRNA comprises a sequence encoding the SARS-CoV-2 omicron variant spike protein B.1.1.529 and a VEEV replicon mRNA backbone (SEQ ID NO: 31). In a second example, the mRNA comprises a sequence encoding the SARS-CoV-2 spike B.1.351-PP-D614G RNA sequence and a VEEV replicon mRNA backbone (SEQ ID NO: 27). [Table 10] [Table 11] Example 14 Self-replicating mRNA construct

[0214] A plasmid encoding the T7 promoter followed by the 5' and 3' UTRs and nonstructural genes of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83 was generated using standard DNA synthesis and cloning methods. The VEEV replicon mRNA backbone is set forth in SEQ ID NO:20. Example 15 Alphavirus-derived replicon RNA vaccine for SARS-CoV-2 neutralizing antibody and T cell responses in non-human primates

[0215] The immunogenicity and safety of the combination of NP-1 to NP-31 and the repRNA-CoV2 S protein vaccine are evaluated in pigtailed macaques. NP-1, consisting of inorganic superparamagnetic iron oxide (SPIO) nanoparticles within a hydrophobic squalene core, is used to enhance the stability of the formulation to protect the RNA replicon from degradation. The replicon RNA (SEQ ID NOs: 1 to 8) is complexed with the NP formulation. Pigtailed macaques were used to test the response to the vaccine. Blood was collected at baseline (week -2 or -1), and 10, 14, 28, and 42 days after prime vaccination. Animals that received the 50 μg vaccine were also bled 10 days after the boost (day 38 after prime). Serum and plasma were collected from whole blood and PBMCs were isolated. Animals were sedated by intramuscular injection of ketamine (Ketaset®; Henry Schein) (10 mg / kg) prior to blood collection or vaccination. 50 μg of vaccine is delivered intramuscularly in the quadriceps in a single 250 μl injection at weeks 0 and 4. To maintain consistency of vaccine formulation and concentration, 250 μg of vaccine is delivered intramuscularly at week 0 by inoculation at five intramuscular injection sites in a 250 μl injection: two in the right quadriceps, one in the left quadriceps, and one each in the left and right deltoids. After injection, all injection sites are monitored for any signs of local reactogenicity. Evaluation of serum chemistry and complete blood counts is performed along with antigen-specific antibody responses.

[0216] Blood is collected from anesthetized monkeys by venipuncture. Antigen-specific IgG, IgG1, IgG2a, and IgG2c responses are detected by enzyme-linked immunosorbent assay (ELISA) using recombinant SARS-CoV-2 S as a capture antigen. SARS-CoV-2 neutralization assays are also performed to determine whether the vaccine composition is able to induce an antibody response against SARS-CoV-2 infection. Immune responses in monkeys are monitored by IFN-γ ELISpot assays and intracellular cytokine staining. Multiparameter flow cytometry is used to determine T cell immune responses using peptide-stimulated PBMCs. S-specific CD4+ or CD8+ T cells are screened before and after immunization. (Example 16) Evaluation of a freeze-dried COVID vaccine in mice

[0217] The following was performed to assay the activity of lyophilized NP-1 and replicon RNA encoding the SARS-CoV-2 spike antigen sequence, the physicochemical properties, potency, and immunogenicity of the reconstituted vaccine: Briefly, the materials in Table 12 were used. [Table 12]

[0218] Preparation of formulation complexes. Lipid nanoparticle / RNA complex compositions were prepared for this assay as shown in Table 13 below. NP-1 or NP-7 were complexed with repRNA at an N:P ratio of 15 to give a final repRNA concentration of 50mg / ml or 100mg / ml ("2x" material), and a sucrose content of 10% or 20% w / v, respectively. The complexed material with 10% sucrose (50mg / ml repRNA) contained 5mM sodium citrate, while the complexed material with 20% sucrose (100mg / ml repRNA) contained 10mM citrate. The complexes were filled into 2ml depyrogenated and baked sterile vials. The complexes with 10% sucrose were filled at 0.7ml per vial, and the complexes with 20% sucrose were filled at 0.35ml per vial. The vials were then either freeze-dried and stored or stored in liquid form. The storage temperature was 25 degrees Celsius or 42 degrees Celsius and was stored for one week. The quantity of freeze-dried and liquid vials per composition is summarized in Table 13. [Table 13]

[0219] Lyophilization Cycle. A SP VirTis Advantage Pro tray and batch lyophilizer with inert gas filling and stoppering capabilities was used. A summary of the lyophilization cycle is shown below in Table 14. Upon completion of the cycle, the vials were back-filled with nitrogen at 48 Torr, stoppered, and then allowed to equilibrate to room pressure. [Table 14]

[0220] Condition Groups. A summary of the 14 groups analyzed in this assay is presented below in Table 15. Groups 1 and 4 were freshly prepared as indicated in the storage column to serve as positive controls for comparison with the standard protocol for vaccine preparation. [Table 15]

[0221] Immunogenicity assay. Induction of anti-spike IgG responses was evaluated in 6-8 week old female C57Bl / 6 mice. Group sizes of 5 mice were used. The schedule is shown in Table 16. [Table 16]

[0222] After one week of storage in a stability chamber at 25 degrees Celsius or 42 degrees Celsius, the lyophilized nanoparticle / RNA complexes were reconstituted in 0.7 ml of sterile milliQ water and gently swirled until no particles were visible to the naked eye. The particle size (z-average) and particle size distribution (PDI) of the complexes were measured. They are summarized in Figure 8, and group designations are shown in Table 15. The particle size and PDI of the freshly prepared NP-1 / WT-S complexes (Group 1) were 76.8 nm and 0.223, respectively. After reconstitution, the lyophilized samples (Groups 7-14) were, on average, 45% (+ / - 11%) larger. A summary of the % change in z-average relative to Group 1 is shown in Table 17. [Table 17]

[0223] Agarose gel electrophoresis of phenol-chloroform extracted repRNA. Storage of liquid formulations of NP-1 / repRNA and NP-7+repRNA in 10% or 20% sucrose for 1 week resulted in partial or complete degradation of the repRNA product (data not shown). Lyophilized preparations of NP-1 / repRNA and NP-7+repRNA in 10% or 20% sucrose were preserved after storage for 1 week as NP-1 / repRNA and NP-7+repRNA (data not shown).

[0224] Potency assay. Storage of lyophilisates of NP-1 / WT-S in 10% sucrose at 25°C for 1 week resulted in dose-dependent expression of spike protein in transfected BHK cells. The expression profile was similar to that of freshly complexed NP-1 / WT-S. Storage of lyophilisates of NP-1 / WT-S in 10% sucrose at 42°C for 1 week significantly reduced protein expression in vitro. Storage of NP-1 / WT-S in 10% sucrose at 25°C or 42°C for 1 week did not produce spike protein in BHK cells (data not shown).

[0225] Anti-D614G spiked IgG responses by ELISA. Serum anti-D614G spiked IgG levels were assessed 14 and 28 days after prime and are shown in Figures 9A and 9B, respectively. Mouse sera were assayed in the anti-D614G spiked ELISA as a 1:40 dilution (day 14) or 1:200 dilution (day 28). Serum IgG levels in μg / ml were interpolated from a four-parameter logistic (4PL) standard curve generated from mouse IgG standards of known concentration.

[0226] Anti-D614G IgG responses 28 days after priming. After 1 week at 25°C, liquid NP-1 / WT-S in 10% sucrose resulted in a statistically significant decrease in anti-spike IgG compared to the freshly prepared NP-1 / WT-S positive control. There was no significant difference in mean IgG levels between freshly prepared NP-1 / WT-S and lyophilized NP-1 / WT-S in 10% sucrose stored at 25°C for 1 week.

[0227] After 1 week at 42°C, lyophilizates of NP-1 / WT-S in 10% sucrose induced 100% seroconversion, but the mean IgG levels were significantly reduced compared to freshly prepared NP-1 / WT-S. A summary of the mean + / - standard deviation IgG concentration data at day 28 post-immunization is shown in Table 18, including p-values ​​determined by ordinary one-way ANOVA compared to freshly prepared NP-1 / WT-S positive control. P<0.05 was considered statistically significant. [Table 18]

[0228] Comparison of freshly prepared and lyophilized formulations. Concentrations of anti-D614G spiked IgG in serum 28 days after priming are shown in FIG. 10. Statistical differences in mean IgG values ​​were determined by ordinary one-way ANOVA and Dunnett's multiple comparison test. All groups were compared to freshly prepared NP-1 / RNA in 10% sucrose. No significant differences were noted between freshly prepared NP-1 / RNA and lyophilized NP-1 / RNA in 10% sucrose stored at 25 degrees Celsius for 7 days. At 42°C, the induction of anti-spiked IgG by lyophilized NP-1 / RNA in 10% or 20% sucrose was significantly lower compared to freshly prepared NP-1 / RNA. The induction of IgG by lyophilized NP-1 / RNA in 20% sucrose stored at 25 degrees Celsius or 42°C was significantly lower than freshly prepared NP-1 / WT-S. The mean IgG induction by lyophilizates of NP-1 / Delta-S in 10% or 20% sucrose stored at 25°C was similar to that of freshly prepared NP-1 / WT-S (not statistically significant). array

[0229] The sequences below (SEQ ID NOs:24-31) are formatted such that the vector backbone and antigen open reading frames are indicated as follows: lowercase letters indicate the VEEV replicon backbone sequence, uppercase letters indicate the spike open reading frame, bold indicates the start codon, and underlines indicate codons that are mutated compared to the parental Wuhan spike sequence.

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[0230] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be used in implementing the present disclosure. It is intended that the scope of the present disclosure be defined by the following claims, and that methods and structures that fall within the scope of these claims and their equivalents are thereby covered.

Claims

1. 1. A dry composition comprising: a) a lipid carrier, said lipid carrier being a nanoemulsion comprising a hydrophobic core and one or more lipids; b) at least one nucleic acid comprising a sequence encoding a sequence capable of expressing an antigen, optionally wherein said antigen is the SARS-CoV-2 spike protein or a functional variant thereof; and c) at least one sugar present in an amount of at least about 50% by weight of said dry composition; A dry composition comprising:

2. The dry composition of claim 1 which is freeze-dried.

3. At about -20 degrees Celsius; At about 25 degrees Celsius; At about 45 degrees Celsius; At about 2 degrees Celsius to about 8 degrees Celsius; or The dry composition of claim 1 , which is thermally stable in these combinations.

4. 10. The dry composition of claim 1, which is thermally stable for at least one week.

5. The dry composition of claim 1 , wherein the hydrophobic core comprises an oil.

6. 6. The dry composition of claim 5, wherein the oil comprises at least one of alpha-tocopherol, lauroyl polyoxylglycerides, monoacylglycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglycerides, vitamin E, caprylic / capric triglyceride, triglyceride esters of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol, dihydroisosqualene (DHIS), farnesene, and squalane.

7. 2. The dry composition of claim 1, wherein the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof.

8. The cationic lipid may be 1,2-dioleoyloxy-3(trimethylammonium)propane, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol, dimethyldioctadecylammonium, 1,2-dimyristoyl-3-trimethylammoniumpropane, dipalmitoyl (C16:0)trimethylammoniumpropane, distearoyltrimethylammoniumpropane, N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium, chloride, N,N-dioleoyl-N,N-dimethyla ammonium chloride, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-3-dimethylammonium-propane, and 1,2-dilinoleyloxy-3-dimethylaminopropane, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), tetrakis(8-methylnonyl)3,3',3",3'''-(((methylazanediyl)bis(propane) ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, (3S,8S,9S ,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol, bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione, 3β-[ N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, 1,2-distearoyl-s n-Glycero-3-phosphocholine, ethylphosphatidylcholine, hexa(octan-3-yl) 9,9',9",9''',9'''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (((3,6-di 8. The dry composition of claim 7, wherein the aryl group is selected from the group consisting of oxopiperazine-2,5-diyl)bis(butane-4,1-diyl)bis(azanetriyl)tetrakis(ethane-2,1-diyl)(9Z,9'Z,9"Z,9'''Z,12Z,12'Z,12"Z,12'''Z)-tetrakis(octadeca-9,12-dienoate), N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide, and combinations thereof.

9. 2. The dry composition of claim 1, wherein the lipid carrier comprises at least one surfactant selected from the group consisting of a hydrophobic surfactant, a hydrophilic surfactant, and any combination thereof.

10. 10. The dry composition of claim 9, wherein the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate, and the hydrophilic surfactant comprises a polysorbate.

11. 2. The dry composition of claim 1, wherein the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm and an average polydispersity index ranging from about 0.1 to about 0.

4.

12. The dry composition of claim 1 , wherein the at least one nucleic acid is DNA, RNA, or self-replicating RNA.

13. 2. The dry composition of claim 1, wherein the at least one sugar is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof.

14. 2. The dry composition of claim 1, wherein the at least one nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NOs: 1-8.

15. The dry composition of claim 1, wherein the at least one sugar is present in an amount of at least about 50 mg.

16. A pharmaceutical composition comprising the dry composition of claim 1 reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier.

17. Use of the dry composition described in claim 1 in the preparation of a medicament for generating an immune response in a subject, characterized in that when the medicament is used, a therapeutically effective amount of the dry composition is reconstituted in a suitable diluent and administered to the subject.

18. A composition comprising: A lipid carrier, comprising: Liquid oil; Cationic lipids; Hydrophilic surfactant; a lipid carrier comprising a hydrophobic surfactant; and at least one nucleic acid, wherein the at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NOs: 1-7.

19. The composition described in claim 18, wherein at least one of the nucleic acids further encodes an RNA polymerase.

20. The composition described in claim 19, wherein the RNA polymerase is Venezuelan equine encephalitis virus (VEEV) RNA polymerase.

21. The composition described in claim 19, wherein at least one nucleic acid encoding the RNA polymerase comprises a nucleic acid sequence of SEQ ID NO:

20.

22. The cationic lipid is 1,2-dioleoyloxy-3(trimethylammonium)propane, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol, dimethyldioctadecylammonium, 1,2-dimyristoyl-3-trimethylammoniumpropane, dipalmitoyl (C16:0)trimethylammoniumpropane, distearoyltrimethylammoniumpropane, N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium, chloride, N,N-dioleoyl-N,N-dimethyl ammonium chloride, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-3-dimethylammonium-propane, 1,2-dilinoleyloxy-3-dimethylaminopropane, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), tetrakis(8-methylnonyl)3,3',3",3'''-(((methylazanediyl)bis(propane) ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, (3S,8S, 9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol, bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate tate, 1,2-distearoyl-sn-glycero-3-phosphocholine, ethylphosphatidylcholine, hexa(octan-3-yl)9,9',9",9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate, heptadecane-9yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino )octanoate, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9"Z,9'''Z,12Z,12'Z,12"Z,12'''Z)-tetrakis(octadeca-9,12-dienoate), or N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide.

23. The composition of claim 18, wherein the liquid oil is alpha-tocopherol, coconut oil, grape seed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E.

24. The composition of claim 18, wherein the lipid carrier comprises a hydrophobic core, inorganic particles, or both.

25. The composition of claim 24, wherein the inorganic particles comprise a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.

26. The composition of claim 25, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.

27. The composition of claim 18, wherein the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate.

28. The composition of claim 18, wherein the hydrophilic surfactant is a polysorbate.

29. A pharmaceutical composition comprising the composition of claim 18 and a pharma- ceutically acceptable carrier.

30. Use of the composition of claim 18 in the preparation of a medicament for generating an immune response in a subject, characterized in that when the medicament is used, a therapeutically effective amount of the composition is administered to the subject.

31. The dry composition of claim 1 for generating an immune response in a subject, characterized in that a therapeutically effective amount of the dry composition is reconstituted in a suitable diluent and administered to the subject.

32. The composition of claim 18 for generating an immune response in a subject, wherein a therapeutically effective amount of the composition is administered to the subject.

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