RNA vaccines against infectious diseases

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 lipid carriers, optionally including inorganic particles, that can be mixed with nucleic acid.Methods of using these compositions as vaccines for treating infectious diseases are also provided.Vaccination can provide preventive protection against infectious diseases, including but not limited to viral, bacterial and / or parasitic diseases.

Description

[Technical field]

[0001] cross reference This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 247,175, filed September 22, 2021, and U.S. Provisional Patent Application No. 63 / 297,498, filed January 7, 2022, the contents of each of which are incorporated by reference herein in their entirety. [Background technology]

[0002] background Vaccination can provide preventative protection against infectious diseases, including but not limited to viral, bacterial and / or parasitic diseases. For example, influenza infection is the seventh leading cause of death in the United States, causing 200,000 hospitalizations and 40,000 deaths in the United States per year, and approximately 3-5 million hospitalizations and 300,000-500,000 deaths per year worldwide. Millions of people receive influenza vaccines annually for protection against seasonal influenza. Vaccination can also rapidly prevent the spread of emerging influenza pandemics. Given the ability of infectious disease agents to develop resistance to vaccines, there is a need for improved efficiency in vaccine production and the development of vaccines with reduced changes evaded by infectious disease agents through evolution. Summary of the Invention [Means for solving the problem]

[0003] A quick overview The following aspects and embodiments thereof, as described and illustrated below, are intended to be exemplary and illustrative, and not limiting in scope.

[0004] 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 encoding an antigenic sequence, the antigenic sequence comprising a viral, bacterial, fungal, or parasitic antigenic sequence, or a sequence encoding a functional variant of any of the foregoing, wherein the viral antigenic sequence is not a SARS-CoV-2 antigenic sequence or a functional variant thereof.

[0005] 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 an antigenic sequence encoding an influenza hemagglutinin protein stem region or a functional variant thereof.

[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 an antigenic sequence encoding a VZV protein 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 an antigenic sequence encoding a respiratory syncytial virus (RSV) antigen or a functional variant thereof.

[0008] 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 oleic acid coated iron oxide nanoparticles, the oleic acid coated iron oxide nanoparticles being of a size ranging from about 5 nanometers (nm) up to 25 nm, optionally wherein the oleic acid coated iron oxide nanoparticles are 12 nm in size; and at least one nucleic acid comprising a sequence at least 85% identical to SEQ ID NOs: 1-6, 38-47.

[0009] 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, and oleic acid coated iron oxide nanoparticles present in an amount of about 0.005 mg; and at least one nucleic acid comprising the sequence of at least SEQ ID NOs: 1-6, 38-47.

[0010] Further provided herein is a composition comprising: (a) a lipid carrier that is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more oils or lipids; and (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, wherein the antigen is an infectious pathogen protein.

[0011] Further provided herein is a dry composition comprising a lipid carrier, which is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, wherein the antigen is an infectious disease protein; and at least one cryoprotectant.

[0012] Further provided herein is a vaccine comprising the composition provided herein. Further provided herein is a vaccine composition comprising: (a) a lipid carrier, which is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, wherein the antigen is an infectious disease protein.

[0013] 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 an infectious disease antigen.

[0014] Further provided herein is a pharmaceutical composition comprising a composition or vaccine composition provided herein; and a pharma- ceutically acceptable excipient.

[0015] 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. 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 an antigen.

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

[0017] Further provided herein is a method of reducing the severity of an infectious disease, comprising administering to a subject prior to infection 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 encoding a sequence capable of expressing an antigen, the antigen being derived from an infectious agent.

[0018] 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.

[0019] Provided herein is a method for treating influenza infection, comprising administering to a subject a composition provided herein, thereby treating influenza infection. Provided herein is a method for treating VZV infection in a subject, comprising administering to a subject a composition provided herein, thereby treating VZV infection. Provided herein is further a method for treating RSV infection in a subject, comprising administering to a subject a composition provided herein, thereby treating RSV infection.

[0020] Further provided herein is a kit comprising the composition provided herein, a package, and materials therefor. Further provided herein is a kit comprising: (a) a lipid carrier, which is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, wherein the antigen is derived from an infectious agent.

[0021] 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 that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0022] [Figure 1-1] 1A-1C show schematic diagrams of exemplary nanoparticle (NP) carriers. Fig. 1A shows an oil-in-water emulsion. Fig. 1B shows a nanostructured lipid carrier (NLC). Fig. 1C shows a nanoparticle with inorganic nanoparticles in liquid oil. [Figure 1-2] Same as above.

[0023] [Diagram 2] FIG. 2 shows RSV-F IgG levels in C57Bl / 6 and BALB / c mice injected IM with 2.5 μg of RSV repRNA formulated with a lipid carrier. Blood was collected 28 days after immunization, and serum was prepared and evaluated by ELISA. Replicon numbers are shown in brackets (645 codes for RSV-G and 646 codes for RSV-F). Each point represents data from an individual animal, whiskers represent minimum to maximum, boxes represent interquartile range, and horizontal bars depict median. X-axis: repRNA RSV antigen. Y-axis: immunoglobulin G (IgG) concentration (micrograms / milliliter).

[0024] [Diagram 3] 3A-3B show anti-G IgG levels in C57Bl / 6 and BALB / c mice injected IM with 2.5 μg of RSV repRNA formulated with a lipid carrier. Blood was collected 28 days after immunization and serum was prepared and evaluated by ELISA. Replicon counts are shown in brackets. Each point represents data from an individual animal, whiskers represent minimum to maximum, boxes represent interquartile range, and horizontal bars depict median. X-axis: repRNA RSV antigen. Y-axis: immunoglobulin G (IgG) concentration (micrograms / milliliter).

[0025] [Figure 4A] Figures 4A-4B show the increase in protein production induced by the Miglyol lipid carrier formulation, NP-3. Figure 4A shows the first assay. Figure 4B shows the second assay. [Figure 4B] Same as above.

[0026] [Figure 5A] Figures 5A-5B show the reduction in immune responses induced by Miglyol lipid carrier formulation, NP-3. Figure 5A shows the first assay. Figure 5B shows the second assay. [Figure 5B]Same as above.

[0027] [Figure 6] Figures 6A-6B show the correlation between improved protein production and lower TNF (e.g., TNFα) stimulation observed with NP-3 as a result of the first and second assays. Figure 6A shows the first assay. Figure 6B shows the second assay.

[0028] [Figure 7A] Figures 7A-7C show the effect of VZV repRNA on antibody titers and expression. Figure 7A shows a schematic of the repRNA encoding the VZV repRNA vaccine constructs - full-length glycoprotein E containing the transmembrane sequence of gE (VZV-FL-gE; black); FL-gI; gE that is N-terminally truncated; full-length gI followed by either a Thosea asigna virus 2A (T2A) ribosome skipping peptide or an internal ribosome entry site (IRES) to accommodate for co-expression of downstream gE; and secreted gE lacking the transmembrane sequence. Figure 7B shows a Western blot, probed with anti-VZV-gE antibody. Figure 7C shows serum antibody titers against soluble VZV gE measured by ELISA 14 days after vaccination. [Figure 7B] Same as above. [Figure 7C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] 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.

[0030] Detailed Description Provided herein is a composition for inducing immune response against infectious microorganisms, its kit, method and use.In brief, further described herein are (1) nanoparticle carrier system; (2) nucleic acid encoding microbial antigen and RNA polymerase; (3) combination composition; (4) heat stable, dry and freeze-dried vaccine; (5) pharmaceutical composition; (6) dosing; (7) administration; (8) therapeutic application; and (9) kit. definition

[0031] 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.

[0032] 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.

[0033] 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."

[0034] 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.

[0035] 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.

[0036] 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.

[0037] As used herein, the term "about" or "approximately" refers to a range of up to ±20% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may 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 stated, the term "about" is implicit and in this context refers to within an acceptable error range of the particular value.

[0038] The term "effective amount" or "therapeutically effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. Nanoparticle Carrier System

[0039] Provided herein are various compositions that include nanoparticles or a multiplicity of nanoparticles.Nanoparticles are also referred to herein as carriers or abbreviated as NP.Provided herein are nanoparticles that can be organic, inorganic, or a combination of inorganic and organic materials with a diameter of less than about 1 micrometer (μm).In some embodiments, provided herein are nanoparticles that are used as a delivery system for provided herein bioactive agents.

[0040] A variety of nanoparticles and nanoparticle formulations (i.e., nanoemulsions) may be used. Exemplary nanoparticles are shown in Figures 1A-1C. 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 nanoparticles (LNPs or SLNs), mixed-phase core NLCs, ionizable 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.

[0041] An oil-in-water emulsion, as shown in FIG. 1A (not to scale), is a stable miscible fluid that contains oil droplets dispersed in a water or aqueous phase. FIG. 1B (not to scale) shows a nanostructured lipid carrier (NLC), which may contain a blend of solid organic lipids (e.g., trimyristin) and liquid oils (e.g., squalene). In an NLC, the solid lipids are dispersed in the liquid oil. The entire nanodroplets are dispersed in the aqueous (water) 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. 1C (not to scale). In that case, the entire nanodroplets are dispersed as colloids in the aqueous (water) phase. In some embodiments, the nanoparticles provided herein are dispersed in an aqueous solution. Non-limiting examples of aqueous solutions include water (e.g., sterile, distilled, deionized, ultrapure, RNAse-free, etc.), saline (e.g., Krebs, Ascaris, Dent's, Tet's saline), or 1% (w / v) dimethyl sulfoxide (DMSO) in water.

[0042] 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-[Ν-(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 (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-(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), 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''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, including PEGylated versions of any of the above lipids (e.g., DSPE-PEG). In some embodiments, the nanoparticles provided herein comprise DOTAP;

[0043] In some embodiments, the nanoparticles provided herein comprise an oil. In some embodiments, the oil is in a liquid phase. Non-limiting examples of oils that can be used include 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. In some embodiments, the nanoparticles provided herein comprise a triglyceride. Exemplary triglycerides include, but are not limited to, capric triglyceride, caprylic triglyceride, caprylic and capric triglycerides, triglyceride esters, and triglycerin myristate. In some embodiments, the oil is in a solid phase. In some embodiments, the oil comprises solanesol.

[0044] 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.

[0045] 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, a metal oxide, a metal hydroxide, or a metal phosphate. In some embodiments, the nanoparticles provided herein comprise a metal salt, but is not limited to, a metal salt, a metal oxide, a metal hydroxide, a metal phosphate, or a metal salt. In some embodiments, the nanoparticles provided herein comprise a metal salt, but is not limited to, a metal salt, a metal oxide, a metal hydroxide, a metal salt ... x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10(PO4)6(OH)2), iron gluconate, or iron sulfate. The inorganic particles can 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 (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3). In some embodiments, the metal is aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid, or a surfactant, such as oleic acid, oleylamine, SDS, TOPO, or DSPA, is used to coat the inorganic solid nanoparticles before mixing with the liquid oil to form a hydrophobic core.

[0046] In some embodiments, the metal may include a paramagnetic, superparamagnetic, ferrimagnetic, or ferromagnetic compound. In some embodiments, the metal is superparamagnetic iron oxide (Fe3O4).

[0047] In some embodiments, the nanoparticles provided herein comprise cationic lipids, oils, 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 lipids, oils, inorganic particles, and surfactants.

[0048] 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 having a hydrophilic-hydrophobic balance (HLB) value of 10 or less, e.g., 5 or less, 1-5, or 4-5. For example, the hydrophobic surfactant can be a sorbitan ester having an HLB value of 1-5 or 4-5. In some embodiments, the nanoparticles provided herein comprise a hydrophilic surfactant, also known as an emulsifier.

[0049] 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. In some embodiments, the nanoparticles or lipid carriers provided herein comprise hydrophilic surfactants. 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.

[0050] The nanoparticles provided herein comprise a hydrophobic core surrounded by a lipid membrane (e.g., a cationic lipid, e.g., DOTAP). In some embodiments, the hydrophobic core comprises one or more inorganic particles; a phosphate-terminated lipid; and a surfactant.

[0051] The inorganic solid nanoparticles described herein may 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 may be coated with hydrophobic molecules (or surfactants) to promote the 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 that is a phosphorus-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant. A typical carboxylate-terminated surfactant includes oleic acid. A typical 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 are coated with a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, before being mixed with the liquid oil to form a hydrophobic core.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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] [Table 1-6] [Table 1-7]

[0056] 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 a medium chain triglyceride. 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 / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol. In some embodiments, the immune stimulant can 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 can increase the total amount of protein produced but decrease the immune response to the compositions provided herein.

[0057] The nanoparticles provided herein can be of various average diameters in terms of size. In some embodiments, the nanoparticles provided herein have an average diameter (z-average hydrodynamic diameter, as 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.

[0058] 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.

[0059] 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, about 0.2:1 to about 3:1, about 0.3:1 to about 2:1, about 0.5:1 to about 2:1, or about 1:1 to about 2:1. 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. In some embodiments, the nanoparticles provided herein comprise a ratio of esters that results in a hydrophilic-lipophilic balance between 8 and 11. 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. In some embodiments, the lipid carrier-nucleic acid complexes are formed via non-covalent interactions or via reversible covalent interactions. Nucleic Acids for Infectious Disease Antigen Expression

[0060] Provided herein are compositions comprising nanoparticles and nucleic acids. In some embodiments, the nucleic acid is in a complex with the nanoparticle. In some embodiments, the nucleic acid is in a complex with the membrane of the nanoparticle. In some embodiments, the nucleic acid is in a complex with the hydrophilic surface of the nanoparticle. In some embodiments, the nucleic acid is within the nanoparticle. In some embodiments, the nucleic acid is within the hydrophobic core.

[0061] In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The nucleic acid may be linear or may contain a secondary structure (e.g., a hairpin). In some embodiments, the nucleic acid is a polynucleotide that contains modified nucleotides or bases and / or their analogs. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the compositions provided herein.Modified nucleobases that may be incorporated into modified nucleosides and nucleotides and may be 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); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine); ms2i6A (2-methylthio- N6 isopentenyladenosine;io6A(N6-(cis-hydroxyisopentenyl)adenosine);ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine);g6A(N6-glycinylcarbamoyladenosine);t6A(N6-threonylcarbamoyladenosine);ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine);m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6-hydroxynorvalylcarbamoyladenosine);ms 2hn6A(2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine);Ar(p)(2'-O-ribosyladenosine (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-formylcytidine);m5Cm(5,2-O-dimethylcytidine);ac4Cm(N4 acetyl 2 TO 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 ester)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW. *(unmodified hydroxywybutosine); imG (wybutosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-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);s2Um(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-methoxycarbonylmethyl-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-trimethylguanosine);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-(C1-C6)-alkyluracil , 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-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. Optionally, the nucleic acid may contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. The RNA sequence may be modified with respect to its codon usage, for example, to increase the translation efficiency and half-life of the RNA. A poly-A tail (e.g., of about 30 or more adenosine residues) may be attached to the 3' end of the RNA to increase its half-life. The 5' end of the RNA may be capped with a modified ribonucleotide or derivative thereof having the structure m7G(5')ppp(5')N (cap 0 structure) (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), which may be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. The cap structure may confer stability and translation efficiency to the RNA molecule. The 5' cap of the 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 translation efficiency. The Cap 1 structure can also increase in vivo potency.

[0062] 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, 50, 75, 100, 150, 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 acid provided herein is 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, 1 mg. In some embodiments, the nucleic acid provided herein is present in an amount of about 5 μg, about 10 μg, about 25 μg, about 50 μg, or about 100 μg. In some embodiments, the nucleic acid provided herein is present in an amount of up to about 5 μg, about 10 μg, about 25 μg, about 50 μg, or 100 μg. 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. infectious disease antigen

[0063] Provided herein are infectious disease antigens for recognition by a host. In some embodiments, the infectious disease antigen is a nucleic acid encoding an antigenic protein sequence. In some embodiments, the compositions provided herein comprise at least one nucleic acid sequence comprising a sequence encoding an antigen derived from a microorganism. In some embodiments, the microorganism is an infectious microorganism. Non-limiting examples of infectious microorganisms and agents include, but are not limited to, viruses, such as adenovirus, herpes simplex 1 virus, herpes simplex 2 virus, encephalitis virus, papilloma virus, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (CMV), chikungunya virus, human herpes virus 8, human papilloma virus (HPV), BK virus, JC virus, smallpox, poliovirus (polio virus), Hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, Coxsackievirus, Hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratory syndrome (SARS) virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, Hepatitis E virus, Human immunodeficiency virus (HIV), Influenza virus (influenza A or B), Guanarito virus, Junin virus, Lassa virus, viruses, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus (RSV), human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D, rotavirus, orbivirus, coltivirus, bannavirus, Zika virus, hantavirus, West Nile virus, Middle East respiratory syndrome (MERS) coronavirus, Japanese encephalitis virus, and eastern equine encephalitis;Bacteria, such as Acetobacter, Acinetobacter, Actinomyces, Agrobacterium, Anaplasma, Azorhizobia, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkkolderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Peptostreptococcus, Porphyromonas, Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Streptococcus pneumoniae, Treponema, Vibrio, Walbachia, and Yersinia; fungi, such as Aspergillus, Saccharomyces, Cryptococcus, Coccidioides, Neurospora, Histoplasma, Blastomyces;Parasites such as Babesia sp., Cryptosporidium sp., Plasmodium sp., Toxoplasma sp. Plasmodium sp., Plasmodium falciparum, Plasmodium vivax, Cryptosporidium parvum, Cryptosporidium hominis, Eimeria sp., Eimeria tenella, Theileria sp., Theileria parva, Toxoplasma sp. Toxoplasma gondii, Trypanosoma brucei subspecies, Trypanosoma cruzi, Leishmania sp., and Leishmania major; and yeasts such as Candida.

[0064] In some embodiments, the antigen is derived from a microorganism that causes severe respiratory disease in mammalian populations, hi some embodiments, the antigen is a surface protein or a transmembrane protein expressed on the surface of a microbial organism.

[0065] In some embodiments, the viral antigen is an influenza virus antigen. In some embodiments, the influenza virus antigen is a hemagglutinin antigen. Hemagglutinin (abbreviated as HA) is a protein present on the surface of influenza viruses. On the viral surface, the hemagglutinin protein exists in a homotrimer state, with each monomer of the homopolymer being composed of two subunits, HA1 and HA2, linked by disulfide bonds. Structurally, the hemagglutinin protein is composed of several domains: a globular head domain, a stalk domain (also called stem or stem protein), a transmembrane domain, and a cytoplasmic domain. Generally, during infection of a host cell (e.g., a eukaryotic cell, e.g., a human cell) by influenza virus, the hemagglutinin protein recognizes and binds to the sialic acid of a receptor on the surface of the host cell, thus facilitating the attachment of the virus to the host cell. After viral endocytosis and acidification of the endosome, the hemagglutinin protein undergoes a pH-dependent conformational change that allows it to facilitate fusion of the viral envelope with the host cell endosomal membrane and entry of viral nucleic acid into the host cell. In general, influenza viruses are classified based on the amino acid sequence of the viral hemagglutinin protein and / or the amino acid sequence of the viral neuraminidase (NA). In some embodiments, the nucleic acid provided herein comprises an antigenic sequence encoding an influenza hemagglutinin protein. In some embodiments, the nucleic acid provided herein comprises an antigenic sequence encoding an influenza hemagglutinin protein stem region or a functional variant thereof. In some embodiments, the hemagglutinin antigen is of subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18. The amino acid sequence differences between the different subtypes of hemagglutinin proteins are found primarily within the sequence of the head domain of the protein.The amino acid sequence of the stem region is believed to be highly conserved among hemagglutinin subtypes compared to the sequence of the head domain. In some embodiments, the hemagglutinin antigen does not include the head domain (HA1). In some embodiments, the hemagglutinin antigen includes a portion of the head domain (HA1). In some embodiments, the hemagglutinin antigen does not include the cytoplasmic domain. In some embodiments, the hemagglutinin antigen includes a portion of the cytoplasmic domain. In some embodiments, a truncated hemagglutinin antigen. In some embodiments, the truncated hemagglutinin protein includes a portion of the transmembrane domain. In some embodiments, the truncated hemagglutinin protein includes the stem region or a functional fragment thereof.

[0066] In some embodiments, the viral antigen is a Varicella-Zoster Virus (VZV) antigen. In some embodiments, the VZV antigen is a glycoprotein E (gE) antigen, a glycoprotein B (gB) antigen, a glycoprotein H (gH) antigen, a glycoprotein L (gL) antigen, a glycoprotein N (gN) antigen, or a glycoprotein I (gI) antigen. In some embodiments, the viral antigen is a coronavirus antigen. In some embodiments, the coronavirus is a SARS-CoV-1 coronavirus or a Middle East Respiratory Syndrome (MERS) coronavirus antigen. In some embodiments, the coronavirus antigen is a spike (S) protein antigen. In some embodiments, the viral antigen is an Epstein-Barr Virus (EBV) antigen. In some embodiments, the viral antigen is a Herpes Simplex Virus (HSV) antigen. In some embodiments, the Herpes Simplex Virus antigen is an HSV1 or HSV2 antigen. In some embodiments, the HSV antigen is glycoprotein B, glycoprotein E, glycoprotein L, glycoprotein M, or glycoprotein I. In some embodiments, the viral antigen is a rabies virus antigen. In some embodiments, the rabies virus antigen is a nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) or polymerase (L) antigen. In some embodiments, the viral antigen is a cytomegalovirus (CMV, human beta herpesvirus 5) antigen. In some embodiments, the CMV antigen is glycoprotein B.Non-limiting examples of viral antigens for inclusion include Zika virus envelope protein (ZIKV E), Zika virus precursor membrane and envelope protein (prM-ENV), SARS-CoV2 spike (S) protein and envelope (E) protein, HIV p24 antigen and Nef protein, influenza virus hemagglutinin (HA) antigens (H2, H3, H5, H6, H7, H8 and H9), influenza virus neuraminidase, rubella E1 and E2 antigens, rotavirus VP7sc antigen, RSV Examples include M2 ​​protein, cytomegalovirus envelope glycoprotein B, S, M and L proteins of hepatitis B virus, rabies glycoprotein, rabies nucleoprotein, Crimean-Congo hemorrhagic fever glycoprotein Gc and / or Gn, Nipah-Henipavirus glycoprotein, Hendra virus glycoprotein, human papillomavirus E6 protein, human papillomavirus E7 protein, human papillomavirus L1 protein, or human papillomavirus L2 protein.

[0067] In some embodiments, the antigen is a viral antigen. In some embodiments, the antigen is a respiratory syncytial virus (RSV) antigen. In some embodiments, the antigen is the RSV glycoprotein (G), RSV-G. In some embodiments, the antigen is the RSV fusion (F) glycoprotein, RSV-F. In some embodiments, the antigen is a Zika virus antigen. In some embodiments, the Zika virus antigen is an envelope (E) protein.

[0068] In some embodiments, the antigen is a bacterial antigen. In some embodiments, the bacterial antigen is a Mycobacterium tuberculosis antigen. In some embodiments, the Mycobacterium tuberculosis antigen is H37Rv, malate synthase, or MPT51. In some embodiments, the bacterial antigen is a Chlamydia trachomatis antigen. In some embodiments, the Chlamydia trachomatis antigen is a major outer membrane protein antigen. In some embodiments, the bacterial antigen is a Staphylococcus aureus antigen.

[0069] In some embodiments, the nucleic acids provided herein encode an antigen or fragment thereof listed in Table 2. In some embodiments, the nucleic acids comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence that specifically binds to an antigen listed in Table 2. 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 2. The percent sequence identity (%) for a given sequence compared 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. The percent identity can be calculated using alignment methods known in the art, for example, alignment of sequences, which can be performed using publicly available software such as BLAST, Align, ClustalW2, etc. One of skill in the art can determine appropriate parameters for alignment, however, the default parameters for BLAST are specifically contemplated. Exemplary nucleic acid sequences encoding exemplary viral and bacterial antigens are listed in Table 2. [Table 2-1] [Table 2-2]

[0070] In some embodiments, the compositions provided herein comprise a nucleic acid sequence comprising any one of SEQ ID NOs: 1-6. In some embodiments, the compositions provided herein comprise a nucleic acid sequence encoding an amino acid sequence comprising any one of SEQ ID NOs: 7-33. In some embodiments, the compositions provided herein comprise a DNA sequence complementary to any one of SEQ ID NOs: 1-6. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO: 7. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO: 8. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO: 9. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO: 10. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO: 11. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO: 12. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NOs: 13-18. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NOs: 20-21. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding any one of the antigens of SEQ ID NO:22-27. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO:28. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO:29. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO:30 or 31. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO:32. In some embodiments, the nucleic acid sequence comprises an RNA or DNA sequence encoding an antigen of SEQ ID NO:33. Further provided herein is a composition comprising a nucleic acid sequence encoding a VZV gE protein having a truncated N-terminal domain. Further provided herein is a composition wherein the nucleic acid sequence encoding a VZV gE protein truncated sequence comprises a sequence at least 85% identical to SEQ ID NO:40.

[0071] In some embodiments, the antigen is a parasitic antigen. In some embodiments, the parasitic antigen is a Giardia lamblia antigen, a Leishmaniasis antigen, a Plasmodium falciparum antigen, a Toxoplasma gondii antigen, a Trichomonas vaginalis antigen, a Trypanosoma brucei antigen, a Trypanosoma cruzi antigen, a Schistosoma antigen, a Toxocara antigen, a Trichinella antigen, or a Babesia antigen.

[0072] In some embodiments, the antigen is a fungal antigen. In some embodiments, the fungal antigen is a Cryptococcus antigen, an Aspergillus antigen, a Coccidioides immitis antigen, a Coccidioides posadasii antigen, a Histoplasma capsulatum antigen, a Blastomyces dermatitidis antigen, a Pneumocystis jirovecii antigen, a Trichophyton antigen, a Microsporum antigen, or an Epidermophyton antigen. In some embodiments, the antigen is a yeast antigen. In some embodiments, the yeast antigen is a Candida antigen. self-replicating nucleic acids

[0073] Compositions comprising self-replicating nucleic acids are provided herein. The antigens or fragments 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 non-structural viral proteins, and also comprises 5'-end and 3'-end cis-active replication sequences, and antigen sequences encoding antigenic proteins. The self-replicating nucleotide sequence can include a subgenomic promoter that directs the expression of a heterologous sequence. If necessary, the heterologous sequence can be fused in frame with other coding regions in the self-replicating RNA and / or under the control of an internal ribosome entry site (IRES).

[0074] 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 that code for structural proteins that are necessary for the production of virus particles in the self-replicating RNA. 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 can be designed to induce the production of infectious virus particles that are attenuated or virulent, or to generate virus particles that are capable of a single round of subsequent infection.

[0075] When a self-replicating RNA molecule is delivered to a vertebrate cell, it can produce multiple daughter RNAs by transcription from itself (or from its own antisense copy), even if it is not accompanied by any protein. The self-replicating RNA can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase, which then produces transcripts from the delivered RNA. In this way, the delivered RNA produces multiple daughter RNAs. These transcripts are antisense to the delivered RNA, and can themselves be translated to produce the in situ expression of the encoded antigen, or can be transcribed to produce additional transcripts of the same sense as the delivered RNA, which are translated to produce the in situ expression of the encoded antigen.

[0076] The self-replicating RNA molecules provided herein can contain one or more modified nucleotides, and therefore have improved stability, resistance to in vivo degradation and clearance, and other advantages. In some embodiments, the self-replicating RNA molecules containing modified nucleotides avoid or reduce the stimulation of endosomes and cytoplasmic immune receptors when the self-replicating RNA is delivered to cells. This allows self-replication, amplification, and expression of proteins to occur. This also reduces safety concerns compared to self-replicating RNAs that do not contain modified nucleotides, since the self-replicating RNAs containing modified nucleotides reduce the activation of the innate immune system and the subsequent undesirable consequences (e.g., inflammation at the injection site, irritation at the injection site, pain, etc.). The RNA molecules generated as a result of self-replication are recognized as foreign nucleic acids by cytoplasmic immune receptors. Therefore, the self-replicating RNA molecules containing modified nucleotides provide efficient amplification of RNA in host cells and expression of the antigens provided herein, as well as adjuvant effects.

[0077] 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, the 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 one or more positions, respectively). Preferably, the self-replicating RNA molecule includes modified nucleotides that contain modifications on or within the nitrogenous base but do not contain modified sugar or phosphate moieties. In some examples, between 0.001% and 99% or 100% of the nucleotides 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 the self-replicating RNA molecule are modified nucleotides. In other examples, between 0.001%-99% or 100% of the specific unmodified nucleotides in the self-replicating RNA molecule are replaced with modified nucleotides. For example, about 1% of the nucleotides in the self-replicating RNA molecule that contain uridine may be modified, for example, by replacing uridine with pseudouridine. In other examples, a desired amount (percentage) of two, three, or four specific nucleotides (nucleotides containing 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 specific nucleotides in the self-replicating RNA molecule are modified nucleotides. In other examples, between 0.001% and 20%, between 0.001% and 15%, between 0.001% and 10%, between 0.01% and 20%, between 0.01% and 15%, between 0.1% and 25, between 0.01% and 10%, between 1% and 20%, between 1% and 15%, between 1% and 10%, or about 5%, about 10%, about 15%, about 20% of a particular nucleotide in a self-replicating RNA molecule are modified nucleotides.It is preferred that less than 100% of the nucleotides in a self-replicating RNA molecule are modified nucleotides. It is also preferred that less than 100% of a particular nucleotide in a self-replicating RNA molecule is modified nucleotide. Thus, preferred self-replicating RNA molecules contain at least some unmodified nucleotides.

[0078] Self-replicating RNA molecules containing at least one modified nucleotide can be prepared using any suitable method. Some suitable methods are known in the art for generating RNA molecules containing modified nucleotides. 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 activity of the selected polymerase, such as suitable buffers and suitable salts. The incorporation of nucleotide analogs into self-replicating RNA can be manipulated, for example, to modify the stability of such RNA molecules, increase resistance to RNase, establish replication after introduction into suitable host cells (the "infectivity" of RNA), and / or 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 that contain one or more modified nucleotides.

[0079] Nucleic acid synthesis can also be performed using suitable recombinant methods well known in the art and conventional, including cloning, processing and / or expression of polynucleotides and gene products encoded by such polynucleotides. Random fragmentation and DNA shuffling by PCR reassembly of gene fragments and synthetic polynucleotides are examples of known techniques that can be used to design and manipulate 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, change codon preferences, generate splice variants, introduce mutations, etc.

[0080] 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.

[0081] The composition provided herein comprises self-replicating RNA.Self-replicating RNA (also called replicon) comprises any genetic element, such as plasmid, cosmid, bacmid, phage or virus, that can replicate mainly under its own control.Self-replication provides a system for the self-replication 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.

[0082] RNA polymerases provided herein can include, but are not limited to, alphavirus RNA polymerase, Eastern Equine Encephalitis Virus (EEEV) RNA polymerase, Western 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:34. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 90% identity to the nucleic acid sequence of SEQ ID NO:34. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 95% identity to the nucleic acid sequence of SEQ ID NO:34. In some embodiments, the nucleic acid encoding the RNA polymerase comprises at least 99% identity to the nucleic acid sequence of SEQ ID NO:34. In some embodiments, the nucleic acid encoding the RNA polymerase is SEQ ID NO:34.

[0083] In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 85% identity to RELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGP (SEQ ID NO:35), TQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTE (SEQ ID NO:36), or SEQ ID NO:37. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 90% identity to SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 95% identity to SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises at least 99% identity to SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the amino acid sequence of the VEEV RNA polymerase comprises SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0084] Provided herein are compositions and methods comprising a replicon RNA (repRNA) encoding 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 from Thosea asigna virus (T2A). Combination Compositions

[0085] 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 number of nanoparticles as provided herein. In some embodiments, the nucleic acid further encodes a self-replicating RNA polymerase. In some embodiments, the nucleic acid further encodes a self-replicating RNA-dependent RNA polymerase. In some embodiments, the nucleic acid encoding the self-replicating RNA polymerase is on the same nucleic acid strand (e.g., in cis) as the nucleic acid sequence encoding the protein. In some embodiments, the nucleic acid encoding the self-replicating RNA polymerase is on a different nucleic acid strand (e.g., in trans) than the nucleic acid sequence encoding the protein. In some embodiments, the nucleic acid encoding the self-replicating RNA polymerase is a DNA molecule. In some embodiments, the nucleic acid sequence encoding the antigen provided herein is a DNA or RNA molecule. In some embodiments, the antigen provided herein is encoded by DNA. Nanoparticles for inclusion include, but are not limited to, any one of NP-1 through NP-30, or any one of NP-1 through NP-31. In some embodiments, the nanoparticle comprises NP-1. In some embodiments, the nanoparticle comprises NP-2. In some embodiments, the nanoparticle comprises 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. In some embodiments, nucleic acids for inclusion include, but are not limited to, those that include a region that includes any one or a plurality of SEQ ID NOs: 1-6, 38-54, or those that encode the amino acid sequence of any one of SEQ ID NOs: 7-33. In some embodiments, nucleic acids for inclusion include, but are not limited to, those that include a region that is complementary to any one of SEQ ID NOs: 1-6, 38-54, or those that encode the amino acid sequence of any one of SEQ ID NOs: 7-33. In some instances, the nucleic acid further includes a region that encodes an RNA polymerase, e.g., a region that includes the sequence of SEQ ID NO: 34.

[0086] 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:38, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:39, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:40, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:41, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 42, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 43, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 44, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 45, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 46, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 47, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 48, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 49, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 50, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO: 51, or a functional fragment thereof.In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 52, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 53, or a functional fragment thereof. In some embodiments, the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 54, 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 that encodes an RNA-dependent RNA polymerase. In some embodiments, the RNA-dependent RNA polymerase is VEEV RNA polymerase. In some embodiments, the two nucleic acid coding elements are present in separate nucleic acids. In some embodiments, the two nucleic acid coding elements are present on the same nucleic acid.

[0087] 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:38, 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: 39, 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: 40, 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: 41, 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: 42, 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: 43, 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: 44, 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: 45, 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: 46, 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: 47, 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: 48, 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: 49, 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: 50, 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: 51, 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: 52, 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: 53, or a functional fragment thereof.

[0088] 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:38, 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: 39, 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: 40, 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: 41, 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: 42, 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: 43, 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: 44, 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: 45, 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: 46, 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: 47, 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: 48, 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: 49, 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: 50, 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: 51, 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: 52, 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: 53, or a functional fragment thereof.

[0089] 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:38, 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:39 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:40 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:41 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:42 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:43 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:44 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: 45, 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: 46, 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: 47, 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: 48, 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: 49, 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: 50, 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: 51, 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: 52, 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: 53, or a functional fragment thereof.

[0090] 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 some 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 some embodiments, the N:P molar ratio of the nanoemulsion composition is about 15: 1. In some embodiments, the nanoparticles comprise a nucleic acid provided herein covalently attached to a membrane.

[0091] 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:cationic lipid, hydrophobic surfactant, and hydrophilic 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 is in the range of about 0.1:1 to about 20:1, about 0.5:1 to about 12:1, about 0.5:1 to about 9:1, about 0.5:1 to about 5:1, about 0.5:1 to about 3:1, or 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, at least about 0.7:1. In some embodiments, the molar ratio of oil to surfactant is at least about 0.4:1 and up to 1:1.

[0092] The compositions provided herein may be characterized by the ratio of hydrophilic surfactant to cationic lipid. In some embodiments, the ratio of hydrophilic surfactant to cationic 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. In some embodiments, the cationic lipid is DOTAP.

[0093] Further 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 dehydrated and freeze-dried infectious disease vaccines

[0094] Provided herein are dry or lyophilized compositions and vaccines.Further provided herein are pharmaceutical compositions comprising the dry or lyophilized compositions provided herein, which are 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.

[0095] In some embodiments, lyophilized compositions are made by a low-temperature dehydration process that involves freezing the composition, followed by 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 vaccines provided herein are spray-dried. Spray-drying is a process by which a solution is fed through an atomizer to create a spray, which is then exposed to a heated gas stream to promote rapid evaporation. Once a sufficient liquid mass has evaporated, the remaining solid material in the droplets forms particles that are then separated from the gas stream (e.g., using a filter or cyclone). Drying helps the compositions and vaccines provided herein to be stored at higher temperatures (e.g., above 4°C) compared to the subzero temperatures required for storage of existing mRNA vaccines. In some embodiments, the dry and lyophilized compositions provided herein include: (a) a lipid carrier that is a nanoemulsion comprising (i) a hydrophobic core, (ii) one or more inorganic nanoparticles, and (iii) 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, sugars, proteins, and carbohydrates. In some embodiments, the compositions and methods provided herein include at least one cryoprotectant. Exemplary cryoprotectants for inclusion include, but are not limited to, sucrose, maltose, trehalose, mannitol, or glucose, and any combination thereof.In some embodiments, the additional or alternative cryoprotectant for inclusion is sorbitol, ribitol, erythritol, threitol, ethylene glycol, or fructose. In some embodiments, the additional or alternative cryoprotectant for inclusion is dimethylsulfoxide (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. 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 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, the 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, the sucrose is present in an amount of about 50 to about 500 mg. In some embodiments, the sucrose is present in an amount of about 200 to about 300 mg. In some embodiments, the sucrose is present in an amount of about 250 mg. In some embodiments, sucrose is present in an amount of at least 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.

[0096] 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.

[0097] In some embodiments, the compositions and vaccines 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 vaccines provided herein are thermally stable at about 25°C or standard room temperature. In some embodiments, the compositions and vaccines provided herein are thermally stable at about 45°C. In some embodiments, the compositions and vaccines provided herein are thermally stable at about -20°C. In some embodiments, the compositions and vaccines provided herein are thermally stable at about 2°C to about 8°C. In some embodiments, the compositions and vaccines 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 vaccines 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, and up to 3 months. In some embodiments, the compositions and vaccines 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, and up to 3 months. In some embodiments, the compositions and vaccines 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, and up to 3 months.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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

[0102] 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.Pharmaceutically acceptable 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.

[0103] In some embodiments, the pharmaceutical composition is a suspension comprising the composition provided herein. In some embodiments, the suspension provided herein comprises a multiplicity of nanoparticles or compositions provided herein. In some embodiments, the composition provided herein is in a suspension, optionally a homogenous suspension. In some embodiments, the composition provided herein is in the form of an emulsion.

[0104] 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.

[0105] 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

[0106] The compositions provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage. A dosage unit form is a physically discrete unit of the compositions provided herein appropriate for the subject being treated. However, it will be understood that the total amount of the compositions provided herein to be used will be determined by the attending physician within the scope of sound medical judgment. For any compositions provided herein, the therapeutically effective amount can be estimated first in cell culture assays or animal models such as mice, rabbits, dogs, pigs, or non-human primates. The animal models are also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The therapeutic efficacy and toxicity of the compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED 50 (the dose that is 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, which is the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50 micrograms (μg) or more. Pharmaceutical compositions that exhibit a large therapeutic index can be useful in some embodiments. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for human use. Exemplary total nucleic acid amounts for incorporation into the compositions described herein include about 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50 micrograms (μg) or more. Administration

[0107] 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.

[0108] 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, sublingual, oral 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.

[0109] 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.

[0110] 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

[0111] Provided herein is a method for treating a disease or disorder in a subject. Provided herein is a method for generating an immune response in a subject against an infectious microorganism. In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious disease is a viral infection, a bacterial infection, a parasitic infection, a fungal infection, or a yeast infection.

[0112] In some embodiments, the subject has, is suspected of, or is at risk of developing a viral infection. In some embodiments, the viral infection is an RSV infection, a CMV infection, SARS, rabies, an HPV infection, chicken pox, shingles, a herpes simplex 1 infection, a herpes simplex 2 infection, or influenza. In some embodiments, the subject has, is suspected of, or is at risk of developing a bacterial infection. In some embodiments, the bacterial infection is tuberculosis, chlamydia, gonorrhea, streptococcal pharyngitis, or a Staphylococcus aureus infection. In some embodiments, the subject has, or is suspected of having, a fungal infection. In some embodiments, the subject has, or is suspected of having, a parasitic infection. In some embodiments, the parasitic infection is malaria. In some embodiments, the subject is at risk of developing an infectious disease or disorder. In some embodiments, the subject contracted the infectious disease by contact with another infected subject. In some embodiments, the subject contracted the infectious disease from contaminated drinking water. In some embodiments, the subject is afflicted with an infectious disease from a different species carrying the microorganism. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. kit

[0113] Provided herein is a kit comprising the compositions provided herein, the pharmaceutical compositions provided herein, and, optionally, a delivery system for administration to a subject. 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 can be accessed to remove the 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. In some embodiments, the kit comprises a lipid carrier comprising a liquid oil and a surfactant, wherein the surfactant comprises a cationic lipid, a hydrophilic surfactant and a hydrophobic surfactant; and at least one nucleic acid, wherein the nucleic acid comprises a sequence encoding a viral, bacterial, fungal or parasitic antigen sequence, or a functional variant thereof, wherein the viral antigen sequence is not derived from SARS-CoV-2. Exemplary embodiments

[0114] 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; at least one nucleic acid encoding an antigenic sequence, the antigenic sequence comprising a viral antigenic sequence, a bacterial antigenic sequence, a fungal antigenic sequence, or a sequence encoding a functional variant of any of the foregoing, the viral antigenic sequence being not a SARS-CoV-2 antigenic sequence or a functional variant thereof. Further provided herein is a composition, the viral antigenic sequence is derived from influenza virus, varicella-zoster virus (VZV), RSV, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), human gammaherpesvirus 4, human papillomavirus (HPV), rabies virus, human alphaherpesvirus 1, human immunodeficiency virus (HIV), or human alphaherpesvirus 2. Further provided herein is a composition in which the bacterial antigenic sequence is derived from a Mycobacterium, Streptococcus, Pseudomonas, Salmonella, Staphylococcus, Neisseria, Clostridium, or Chlamydia fungus. Further provided herein is a composition in which the fungal antigenic sequence is derived from an Aspergillus, Saccharomyces, Cryptococcus, Coccidioides, Neurospora, Histoplasma, or Blastomyces fungus. Further provided herein is a composition in which the parasitic antigenic sequence is derived from a Plasmodium parasite. Further provided herein is a composition in which at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NOs: 1-6, 43-47. Further provided herein is a composition in which at least one nucleic acid comprises a sequence at least 90% identical to SEQ ID NOs: 1-6, 43-47. Further provided herein are compositions, wherein at least one nucleic acid comprises a sequence at least 95% identical to SEQ ID NOs: 1-6, 43-47.Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence selected from any one of SEQ ID NOs: 1-6, 43-47. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 1. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 2. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 3. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 4. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 5. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 6. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 43. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO: 44. Further provided herein is a composition wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:45. Further provided herein is a composition wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:46. Further provided herein is a composition wherein at least one nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:47. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:1. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:2. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:3. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:4. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:5. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:6. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:43. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:44. Further provided herein is a composition wherein at least one nucleic acid comprises SEQ ID NO:45.Further provided herein is a composition, wherein at least one nucleic acid comprises SEQ ID NO: 46. Further provided herein is a composition, wherein at least one nucleic acid comprises SEQ ID NO: 47. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence encoding an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 7-33. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence encoding an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 7-33. Further provided herein is a composition, wherein at least one nucleic acid comprises a sequence encoding an amino acid sequence of any one of SEQ ID NOs: 7-33. Further provided herein is a composition, wherein the nucleic acid is in a complex with a lipid carrier. 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: 34. 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. Further provided herein is a composition in which the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, triglyceride ester, or triglycerin myristate. Further provided herein is a composition in which the lipid carrier comprises a hydrophobic core. Further provided herein is a composition in which the lipid carrier comprises inorganic particles. Further provided herein is a composition in which the inorganic particles are within the hydrophobic core. Further provided herein is a composition, wherein the inorganic particles comprise a metal. Further provided herein is a composition, wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.Further provided herein is a composition in which the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide. Further provided herein is a composition in which the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate. Further provided herein is a composition in which the hydrophilic surfactant is a polysorbate. 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 in which the lipid carrier comprises a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to 0.4. Further provided herein is a composition in which the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50, or about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein is a composition in the form of a suspension. Further provided herein is a composition that is lyophilized.

[0115] 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 an antigenic sequence encoding an influenza hemagglutinin protein stem region, or a functional variant thereof. Further provided herein is a composition, the nucleic acid comprising a sequence at least 85% identical to SEQ ID NO:3, or a functional fragment thereof. Further provided herein is a composition, the nucleic acid comprising a sequence at least 90% identical to SEQ ID NO:3, or a functional fragment thereof. Further provided herein is a composition, the nucleic acid comprising a sequence at least 95% identical to SEQ ID NO:3, or a functional fragment thereof. Further provided herein is a composition, the nucleic acid comprising SEQ ID NO:3. Further provided herein is a composition, the nucleic acid encoding an amino acid sequence at least 85% identical to any one of SEQ ID NOs:10-12. Further provided herein is a composition, the nucleic acid encoding an amino acid sequence at least 90% identical to any one of SEQ ID NOs:10-12. Further provided herein is a composition, wherein the nucleic acid encodes an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 10-12. Further provided herein is a composition, wherein the nucleic acid encodes an amino acid sequence comprising any one of SEQ ID NOs: 10-12. Further provided herein is a composition, wherein the nucleic acid is in a complex with a lipid carrier. 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: 34. Further provided herein are compositions 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, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E.Further provided herein is a composition in which the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, triglyceride ester, or triglyceryl myristate. Further provided herein is a composition in which the lipid carrier comprises a hydrophobic core. Further provided herein is a composition in which the lipid carrier comprises inorganic particles. Further provided herein is a composition in which the inorganic particles are within the hydrophobic core. Further provided herein is a composition in which the inorganic particles comprise a metal. Further provided herein is a composition in which the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. Further provided herein is a composition in which the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide. Further provided herein is a composition in which the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate. Further provided herein is a composition in which the hydrophilic surfactant is a polysorbate. 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 in which the lipid carrier comprises a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to 0.4. Further provided herein is a composition in which the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50, or about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein is a composition in the form of a suspension. Further provided herein is a composition that is lyophilized.

[0116] 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 an antigenic sequence encoding a VZV protein or a functional variant thereof. Further provided herein is a composition, the VZV protein or functional variant thereof is glycoprotein E (gE), gI, gB, gH, gL, gN, or a functional fragment thereof. Further provided herein is a composition, the antigenic sequence encoding a VZV protein is at least 85% identical to any one of SEQ ID NOs: 4, 5, 38-43. Further provided herein is a composition, the antigenic sequence encoding a VZV protein is at least 90% identical to any one of SEQ ID NOs: 4, 5, 38-43. Further provided herein is a composition, the antigenic sequence encoding a VZV protein is at least 95% identical to any one of SEQ ID NOs: 4, 5, 38-43. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein comprises any one of SEQ ID NOs: 4, 5, 38-43. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 85% identical to SEQ ID NO: 4. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 90% identical to SEQ ID NO: 4. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 95% identical to SEQ ID NO: 4. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein comprises SEQ ID NO: 4. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 85% identical to SEQ ID NO: 5. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 90% identical to SEQ ID NO: 5. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 95% identical to SEQ ID NO: 5. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein comprises SEQ ID NO: 5.Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 85% identical to SEQ ID NO: 40. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 90% identical to SEQ ID NO: 40. Further provided herein is a composition, wherein the antigen sequence encoding the VZV protein is at least 95% identical to SEQ ID NO: 40. Further provided herein is a composition, wherein the nucleic acid sequence encoding the VZV protein antigen comprises SEQ ID NO: 40. Further provided herein is a composition, wherein the nucleic acid comprises a nucleic acid sequence encoding a VZV gE protein having a truncated N-terminal domain. Further provided herein is a composition, wherein the nucleic acid is in a complex with a lipid carrier. 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: 34. 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. Further provided herein is a composition in which the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, triglyceride ester, or triglycerin myristate. Further provided herein is a composition in which the lipid carrier comprises a hydrophobic core. Further provided herein is a composition in which the lipid carrier comprises inorganic particles. Further provided herein is a composition in which the inorganic particles are within the hydrophobic core. Further provided herein is a composition in which the inorganic particles comprise a metal. Further provided herein are compositions wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.Further provided herein is a composition in which the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide. Further provided herein is a composition in which the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate. Further provided herein is a composition in which the hydrophilic surfactant is a polysorbate. 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 in which the lipid carrier comprises a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to 0.4. Further provided herein is a composition in which the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50, or about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein is a composition in the form of a suspension. Further provided herein is a composition that is lyophilized.

[0117] 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 an antigen sequence encoding a respiratory syncytial virus (RSV) antigen or a functional variant thereof. Further provided herein is a composition, the respiratory syncytial virus antigen is a RSV-F antigen. Further provided herein is a composition, the respiratory syncytial virus antigen is a RSV-G antigen. Further provided herein is a composition, the antigen sequence encoding the respiratory syncytial virus antigen is at least 85% identical to any one of SEQ ID NOs: 1, 2, 45, or 46. Further provided herein is a composition, the antigen sequence encoding the respiratory syncytial virus antigen is at least 90% identical to any one of SEQ ID NOs: 1, 2, 45, or 46. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 95% identical to any one of SEQ ID NO: 1, 2, 45 or 46. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen comprises any one of SEQ ID NO: 1, 2, 45 or 46. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 85% identical to SEQ ID NO: 1. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 90% identical to SEQ ID NO: 1. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 95% identical to SEQ ID NO: 1. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen comprises SEQ ID NO: 1. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 85% identical to SEQ ID NO: 2. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 90% identical to SEQ ID NO: 2. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 95% identical to SEQ ID NO: 2.Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen comprises SEQ ID NO:2. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 85% identical to SEQ ID NO:45. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 90% identical to SEQ ID NO:45. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 95% identical to SEQ ID NO:45. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen comprises SEQ ID NO:45. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 85% identical to SEQ ID NO:46. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 90% identical to SEQ ID NO:46. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen is at least 95% identical to SEQ ID NO:46. Further provided herein is a composition, wherein the antigen sequence encoding the respiratory syncytial virus antigen comprises SEQ ID NO: 46. Further provided herein is a composition, wherein the nucleic acid is in a complex with a lipid carrier. 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: 34. Further provided herein is a composition, wherein 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. Further provided herein are compositions wherein the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglyceride, a triglyceride ester, or triglyceryl myristate.Further provided herein is a composition in which the lipid carrier comprises a hydrophobic core. Further provided herein is a composition in which the lipid carrier comprises an inorganic particle. Further provided herein is a composition in which the inorganic particle is within the hydrophobic core. Further provided herein is a composition in which the inorganic particle comprises a metal. Further provided herein is a composition in which the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. Further provided herein is a composition in which the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide. Further provided herein is a composition in which the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate. Further provided herein is a composition in which the hydrophilic surfactant is a polysorbate. Further provided herein are compositions 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 are compositions in which the lipid carrier comprises a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm with an average polydispersity index ranging from about 0.1 to 0.4. Further provided herein are compositions in which the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Further provided herein are compositions in which the at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50 or about 100 micrograms (μg). Further provided herein are compositions in which the at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein are compositions in the form of a suspension. Further provided herein are compositions that are lyophilized.

[0118] 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 oleic acid coated iron oxide nanoparticles, the oleic acid coated iron oxide nanoparticles being of a size range of about 5 nanometers up to 25 nm; and at least one nucleic acid comprising a sequence at least 85% identical to SEQ ID NOs: 1-6, 38-47. Further provided herein is a composition wherein the oleic acid coated iron oxide nanoparticles are of a size range of about 5 to about 25 nanometers. Further provided herein is a composition wherein the oleic acid coated iron oxide nanoparticles are 12 nanometers in size. Further provided herein is a composition comprising sucrose. Further provided herein is a composition wherein the sucrose is present at about 50 mg. Further provided herein is a composition in which the nucleic acid is in a complex 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:34. Further provided herein is a composition in which the lipid carrier comprises a hydrophobic core. Further provided herein is a composition in which oleic acid coated iron oxide nanoparticles are within the hydrophobic core. 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 in which the lipid carrier comprises a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to 0.4. Further provided herein is a composition, wherein the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg).Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50, or about 100 micrograms (μg). Further provided herein is a composition, wherein at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein is a composition in the form of a suspension. Further provided herein is a composition that is lyophilized.

[0119] 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, and oleic acid coated iron oxide nanoparticles present in an amount of about 0.005 mg; and at least one nucleic acid comprising at least one of the sequences SEQ ID NOs: 1-6, 38-47. Further provided herein is a composition wherein the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Further provided herein is a composition wherein the at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50, or about 100 micrograms (μg). Further provided herein is a composition wherein the at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein is a composition comprising sucrose. Further provided herein is a composition wherein the sucrose is present at about 50 milligrams (mg). Further provided herein is a composition wherein the nucleic acid is in a complex with a lipid carrier. 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:34. Further provided herein is a composition wherein the lipid carrier comprises a hydrophobic core. Further provided herein is a composition wherein oleic acid coated iron oxide nanoparticles are within the hydrophobic core. Further provided herein is a composition wherein 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, wherein the lipid carrier comprises a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to 0.4. Further provided herein is a composition in the form of a suspension.Further provided herein are compositions that are lyophilized.

[0120] Provided herein is a composition comprising: (a) a lipid carrier that is a nanoemulsion comprising a hydrophobic core and one or more inorganic nanoparticles and one or more oils or lipids; (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, wherein the antigen is an infectious pathogen protein. Further provided herein is a composition comprising a nucleic acid comprising a nucleic acid polymerase or encoding a sequence capable of expressing a nucleic acid polymerase. Further provided herein is a composition wherein the nucleic acid sequence is an RNA nucleic acid sequence. Further provided herein is a composition wherein the RNA polymerase or a nucleic acid sequence capable of expressing an RNA polymerase. Further provided herein is a composition comprising a nucleic acid sequence comprising any one of SEQ ID NOs: 1-6, 34-54. Further provided herein is a composition wherein a host cell is capable of expressing an antigen from a nucleic acid sequence, wherein the antigen is derived from an infectious agent. Further provided herein is a composition wherein the infectious agent is a respiratory syncytial virus (RSV). Further provided herein is a composition wherein the antigen comprises the RSV attachment (G) glycoprotein (RSV-G). Further provided herein is a composition, wherein the antigen comprises RSV fusion (F) glycoprotein (RSV-F). Further provided herein is a composition, wherein the infectious agent is not SARS-COV-2. Further provided herein is a composition, wherein the infectious agent is Zika virus. Further provided herein is a composition, wherein the infectious agent is an enterovirus. Further provided herein is a composition, wherein the infectious agent is an influenza virus. Further provided herein is a composition, wherein the hydrophobic core comprises an oil. Further provided herein is a composition, wherein the oil comprises at least one of alpha-tocopherol, lauroyl polyoxylglyceride, monoacylglycerol, propolis, squalene, squalene, miglyol, dehydroisosqualene (DHIS), mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soybean lecithin, triglyceride, vitamin E, and medium chain triglyceride. Further provided herein is a composition, wherein the one or more inorganic nanoparticles are selected from the group consisting of a metal salt, a metal oxide, a metal hydroxide, a metal phosphate, and any combination thereof.Further provided herein is a composition, 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 composition, wherein one or more lipids are cationic lipids. The cationic lipids are selected from the group consisting of 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[Ν-(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 in which the lipid carrier is selected from the group consisting of 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 are compositions in which the lipid carrier has a z-average hydrodynamic diameter in the range of about 40 mm to about 150 nm with an average polydispersity index in the range of about 0.1 to about 0.4. Further provided herein are compositions in which one or more nucleic acids are incorporated into or complexed with the lipid carrier to form lipid carrier-nucleic acid complexes. Further provided herein are compositions in which the lipid carrier-RNA complexes are formed via non-covalent interactions or via reversible covalent interactions. Further provided herein are compositions 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 are compositions that are stable at 2 to 8 degrees Celsius. Further provided herein are compositions in the form of a suspension. Further provided herein are compositions that are lyophilized. Further provided herein are vaccines comprising the compositions provided herein.

[0121] Further provided herein is a vaccine composition comprising: (a) a lipid carrier that is a nanoemulsion comprising a hydrophobic core and one or more inorganic nanoparticles and one or more lipids; (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, wherein the antigen is an infectious disease protein. Further provided herein is a vaccine composition wherein the nucleic acid is RNA. Further provided herein is a vaccine composition further comprising a nucleic acid encoding a polymerase, or a sequence capable of expressing a polymerase. Further provided herein is a vaccine composition further comprising an RNA polymerase, or a nucleic acid sequence capable of expressing an RNA polymerase. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising a sequence set forth in any one of SEQ ID NOs: 1-6 or 34-54. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising SEQ ID NO: 1. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising SEQ ID NO: 2. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising SEQ ID NO: 3. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising SEQ ID NO: 4. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising SEQ ID NO: 5. Further provided herein is a vaccine composition comprising a nucleic acid sequence comprising SEQ ID NO:6. Further provided herein is a vaccine composition comprising a nucleic acid sequence capable of expressing an antigen, wherein the antigen is derived from an infectious agent. Further provided herein is a vaccine composition wherein the infectious agent is a respiratory syncytial virus (RSV). Further provided herein is a vaccine composition wherein the antigen comprises a RSV attachment (G) glycoprotein (RSV-G). Further provided herein is a vaccine composition wherein the antigen comprises a RSV fusion (F) glycoprotein (RSV-F). Further provided herein is a vaccine composition wherein the infectious agent is a Zika virus. Further provided herein is a vaccine composition wherein the infectious agent is an enterovirus. Further provided herein is a vaccine composition wherein the infectious agent is an influenza virus. Further provided herein is a vaccine composition wherein the infectious agent is not 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, and vitamin E, and 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, 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-[Ν-(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, wherein the lipid carrier is selected from the group consisting of: 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (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, wherein the lipid carrier optionally comprises one or more surfactants. Further provided herein is a vaccine composition, wherein 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 in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to about 0.4.Further provided herein are vaccine compositions 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 are vaccine compositions in which the lipid carrier-RNA complex is formed via non-covalent interactions or via reversible covalent interactions. Further provided herein are vaccine compositions 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 are vaccine compositions in which the composition is stable at 2-8 degrees Celsius. Further provided herein are compositions in which the at least one nucleic acid sequence is present in an amount of up to about 100 micrograms (μg). Further provided herein are compositions in which the at least one nucleic acid sequence is present in an amount of up to about 5, about 10, about 25, about 50, or about 100 micrograms (μg). Further provided herein are vaccine compositions in which the at least one nucleic acid sequence is present in an amount of up to about 25 μg. Further provided herein is a vaccine composition in the form of a suspension.Further provided herein is a vaccine composition that is lyophilized.

[0122] Provided herein are compositions and methods for immunoprotecting a subject, comprising administering to a subject a composition provided herein. Further provided herein are compositions for immunoprotecting a subject, comprising: (a) a lipid carrier that is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, the antigen being derived from an infectious agent. Further provided herein are compositions for immunoprotecting a subject, wherein the nucleic acid is RNA. Further provided herein are compositions for immunoprotecting a subject, further comprising a nucleic acid encoding a polymerase, or a sequence capable of expressing a polymerase. Further provided herein are compositions for immunoprotecting a subject, further comprising an RNA polymerase, or a nucleic acid sequence capable of expressing an RNA polymerase. Further provided herein are compositions for immunoprotecting a subject, comprising a nucleic acid sequence comprising a sequence set forth in any one of SEQ ID NOs: 1-6 or 34-54. Further provided herein are compositions for immunoprotecting a subject, comprising a nucleic acid sequence comprising SEQ ID NO: 1. Further provided herein is a composition for immunoprotecting a subject, comprising a nucleic acid sequence comprising SEQ ID NO:2. Further provided herein is a composition for immunoprotecting a subject, comprising a nucleic acid sequence comprising SEQ ID NO:3. Further provided herein is a composition for immunoprotecting a subject, comprising a nucleic acid sequence comprising SEQ ID NO:4. Further provided herein is a composition for immunoprotecting a subject, comprising a nucleic acid sequence comprising SEQ ID NO:5. Further provided herein is a composition for immunoprotecting a subject, comprising a nucleic acid sequence comprising SEQ ID NO:6. Further provided herein is a composition for immunoprotecting a subject, comprising a nucleic acid sequence capable of expressing an antigen, wherein the antigen is derived from an infectious agent. Further provided herein is a composition for immunoprotecting a subject, wherein the infectious agent is respiratory syncytial virus (RSV). Further provided herein is a composition for immunoprotecting a subject, wherein the antigen comprises the RSV attachment (G) glycoprotein (RSV-G).Further provided herein is a composition for immunoprotecting a subject, wherein the antigen comprises a RSV fusion (F) glycoprotein (RSV-F). Further provided herein is a composition for immunoprotecting a subject, wherein the infectious agent is a Zika virus. Further provided herein is a composition for immunoprotecting a subject, wherein the infectious agent is an enterovirus. Further provided herein is a composition for immunoprotecting a subject, wherein the infectious agent is an influenza virus. Further provided herein is a composition for immunoprotecting a subject, wherein the infectious agent is not SARS-CoV-2. Further provided herein is a composition for immunoprotecting a subject, wherein the hydrophobic core comprises an oil. Further provided herein is a composition for immunoprotecting a subject, 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, and vitamin E, and medium chain triglyceride. Further provided herein is a composition for immunoprotecting a subject, wherein 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 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-[Ν-(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, wherein the lipid carrier is selected from the group consisting of: 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (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 for immunoprotecting a subject, wherein the lipid carrier optionally comprises one or more surfactants. Further provided herein is a composition for immunoprotecting a subject, wherein 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 for immunoprotecting a subject, 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 for immunoprotecting a subject, wherein the lipid carrier has a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm, with an average polydispersity index in the range of about 0.1 to about 0.4.Further provided herein is a composition for immunoprotecting a subject, 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 for immunoprotecting a subject, in which the lipid carrier-RNA complex is formed via a non-covalent interaction or via a reversible covalent interaction. Further provided herein is a composition for immunoprotecting a subject, 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 for immunoprotecting a subject, in which the composition is stable at 2-8 degrees Celsius.

[0123] Provided herein is a dry composition comprising: (a) a lipid carrier that is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) at least one nucleic acid sequence encoding a sequence capable of expressing an antigen, the antigen being an infectious disease protein; and at least one cryoprotectant. Further provided herein is a dry composition comprising: a lipid carrier that is a nanoemulsion comprising a hydrophobic core and one or more lipids; one or more nucleic acids; and at least one sugar, wherein the at least one sugar is (i) present in an amount of at least about 50% by weight of the dry composition, or (ii) present 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 about 2 degrees Celsius to 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, monoacylglycerol, 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 squalene. 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 are compositions wherein the one or more lipids comprise a cationic lipid.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[Ν-(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 chloride (DSTAP); 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 in the range of about 40 nm to about 150 nm with an average polydispersity index in the range of 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 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 are compositions 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 are compositions in which the lipid carrier-nucleic acid complex is formed via non-covalent interactions or via reversible covalent interactions. Further provided herein are compositions 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 are compositions 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 are compositions wherein the at least one sugar is present in an amount of at least about 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 mg or more. Further provided herein are compositions wherein the at least one sugar is present in an amount between 50 mg and 250 mg.Further provided herein is a composition in which the at least one sugar is present in an amount of at least about 250 mg. Further provided herein is a composition in which the sugar is present in an amount of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more by weight of the composition. Further provided herein is a composition in which the sugar is present in an amount of 80-98%, optionally 94-96% by weight of the composition. Further provided herein is a composition in which the sugar is present in an amount of about 95% by weight of the composition. Further provided herein is a composition in which the at least one sugar comprises sucrose.

[0124] Provided herein is a pharmaceutical composition comprising the composition or vaccine composition provided herein; and a pharma- ceutically acceptable excipient.Further provided herein is a pharmaceutical composition comprising the dry composition disclosed herein, reconstituted with 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.

[0125] Provided herein is a method of generating an immune response in a subject, comprising administering to the subject a composition provided herein. Provided herein is a method of generating an immune response in a subject, comprising administering to the subject a 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 14 days after the first dose. 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 second dose is administered about 42 days after the first dose. Provided herein is a method in which the second dose is administered about 56 days after the first dose. Provided herein is a method in which the second dose is administered about 70 days after the first dose. Provided herein is a method in which the second dose is administered to the subject, further comprising administering a third dose of the composition. Further provided herein is a method wherein 5 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 10 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 25 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 30 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 35 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 40 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 45 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 50 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 55 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 60 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 65 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 70 micrograms of the composition are administered to the subject.Further provided herein is a method wherein 75 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 80 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 85 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 90 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 95 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 100 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 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. Further provided herein is a method wherein the composition is administered intranasally. Further provided herein is a method wherein the subject has, is suspected of having, or is at risk of developing a viral infection. Further provided herein is a method, wherein the viral infection is influenza infection, VZV infection, RSV infection, severe acute respiratory syndrome (SARS) infection, human gammaherpesvirus 4 infection, human papillomavirus (HPV) infection, rabies virus infection, human alphaherpesvirus 1 infection, or human alphaherpesvirus 2 infection. Further provided herein is a method, wherein the subject has, is suspected of having, or is at risk of developing a bacterial infection. Further provided herein is a method, wherein the bacterial infection is Mycobacterium infection, Streptococcus infection, Pseudomonas infection, Salmonella infection, Staphylococcus infection, Neisseria infection, Clostridium infection, or Chlamydia infection. Further provided herein is a method, wherein the subject has, is suspected of having, or is at risk of developing a parasitic infection, fungal infection, or yeast infection. Further provided herein is a method, wherein the immune response comprises increasing the 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 increasing 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.

[0126] Provided herein is a method of enhancing an immune response in a subject, comprising administering to the subject a composition provided herein, thereby enhancing 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 14 days after the first dose. 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 second dose is administered about 42 days after the first dose. Provided herein is a method in which the second dose is administered about 56 days after the first dose. Provided herein is a method in which the second dose is administered about 70 days after the first dose. Provided herein is a method in which the second dose is administered to the subject, further comprising administering to the subject a third dose of the composition. Provided herein is a method in which 5 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 10 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 25 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 30 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 35 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 40 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 45 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 50 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 55 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 60 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 65 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 70 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 75 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 80 micrograms of the composition is administered to said subject.Further provided herein is a method in which 85 micrograms of the composition are administered to the subject. Further provided herein is a method in which 90 micrograms of the composition are administered to the subject. Further provided herein is a method in which 95 micrograms of the composition are administered to the subject. Further provided herein is a method in which 100 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 in which the mammal is a human. Further provided herein is a method in which the composition is administered intramuscularly. Further provided herein is a method in which the composition is administered intranasally. Further provided herein is a method in which the subject has, is suspected of having, or is at risk of developing a viral infection. Further provided herein is a method in which the viral infection is an influenza infection, a VZV infection, a RSV infection, a severe acute respiratory syndrome (SARS) infection, a human gammaherpesvirus 4 infection, a human papillomavirus (HPV) infection, a rabies virus infection, a human alphaherpesvirus 1 infection, or a human alphaherpesvirus 2 infection. Further provided herein is a method in which the subject has, is suspected of having, or is at risk of developing a bacterial infection. Further provided herein is a method in which the bacterial infection is a Mycobacterium infection, a Streptococcus infection, a Pseudomonas infection, a Salmonella infection, a Staphylococcus infection, a Neisseria infection, a Clostridium infection, or a Chlamydia infection. Further provided herein is a method in which the subject has, is suspected of having, or is at risk of developing a parasitic infection, a fungal infection, or a yeast infection. Further provided herein is a method in which the immune response comprises increasing the titer of neutralizing antibodies against the antigen compared to a subject not receiving the composition. Further provided herein is a method in which the immune response comprises increasing the amount of CD4+ and / or CD8+ positive T cells compared to a subject not receiving the composition. Further provided herein is a method in which the subject is immunocompromised or immunosuppressed.

[0127] Provided herein is a method of treating influenza infection, comprising administering to a subject a composition provided herein, thereby treating influenza infection. 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 14 days after the first dose. 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 second dose is administered about 42 days after the first dose. Further provided herein is a method in which the second dose is administered about 56 days after the first dose. Further provided herein is a method in which the second dose is administered about 70 days after the first dose. Further provided herein is a method further comprising 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 wherein 25 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 30 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 35 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 40 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 45 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 50 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 55 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 60 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 65 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 70 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 75 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 80 micrograms of the composition are administered to the subject.Further provided herein is a method in which 85 micrograms of the composition are administered to the subject. Further provided herein is a method in which 90 micrograms of the composition are administered to the subject. Further provided herein is a method in which 95 micrograms of the composition are administered to the subject. Further provided herein is a method in which 100 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 in which the mammal is a human. Further provided herein is a method in which the composition is administered intramuscularly. Further provided herein is a method in which the composition is administered intranasally. Further provided herein is a method in which the subject has, is suspected of having, or is at risk of developing influenza infection. Further provided herein is a method in which the subject generates an immune response to an antigen. Further provided herein is a method in which the immune response comprises increasing the titer of neutralizing antibodies to the antigen compared to a subject not receiving the composition. Further provided herein is a method in which the immune response comprises increasing the amount of CD4+ and / or CD8+ positive T cells compared to a subject not receiving the composition. Further provided herein are methods, wherein the subject is immunocompromised or immunosuppressed.

[0128] Provided herein is a method of treating a VZV infection in a subject, comprising administering to the subject a composition provided herein, thereby treating the VZV infection. 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 14 days after the first dose. 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 second dose is administered about 42 days after the first dose. Provided herein is a method in which the second dose is administered about 56 days after the first dose. Provided herein is a method in which the second dose is administered about 70 days after the first dose. Provided herein is a method in which the second dose is administered to the subject, further comprising administering to the subject a third dose of the composition. Provided herein is a method in which 5 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 10 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 25 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 30 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 35 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 40 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 45 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 50 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 55 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 60 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 65 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 70 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 75 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 80 micrograms of the composition is administered to said subject.Further provided herein is a method in which 85 micrograms of the composition are administered to the subject. Further provided herein is a method in which 90 micrograms of the composition are administered to the subject. Further provided herein is a method in which 95 micrograms of the composition are administered to the subject. Further provided herein is a method in which 100 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 in which the mammal is a human. Further provided herein is a method in which the composition is administered intramuscularly. Further provided herein is a method in which the composition is administered intranasally. Further provided herein is a method in which the subject has, is suspected of having, or is at risk of developing a VZV infection. Further provided herein is a method in which the subject generates an immune response to an antigen. Further provided herein is a method in which the immune response comprises increasing the titer of neutralizing antibodies to the antigen compared to a subject not receiving the composition. Further provided herein is a method in which the immune response comprises increasing the amount of CD4+ and / or CD8+ positive T cells compared to a subject not receiving the composition. Further provided herein are methods, wherein the subject is immunocompromised or immunosuppressed.

[0129] Provided herein is a method of treating a RSV infection in a subject, comprising administering to the subject a composition provided herein, thereby treating the RSV infection. 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 14 days after the first dose. 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 second dose is administered about 42 days after the first dose. Provided herein is a method in which the second dose is administered about 56 days after the first dose. Provided herein is a method in which the second dose is administered about 70 days after the first dose. Provided herein is a method further comprising administering to the subject a third dose of the composition. Provided herein is a method in which 5 micrograms of the composition are administered to the subject. Provided herein is a method in which 10 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 25 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 30 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 35 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 40 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 45 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 50 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 55 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 60 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 65 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 70 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 75 micrograms of the composition are administered to the subject. Further provided herein is a method wherein 80 micrograms of the composition are administered to the subject.Further provided herein is a method in which 85 micrograms of the composition are administered to the subject. Further provided herein is a method in which 90 micrograms of the composition are administered to the subject. Further provided herein is a method in which 95 micrograms of the composition are administered to the subject. Further provided herein is a method in which 100 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 in which the mammal is a human. Further provided herein is a method in which the composition is administered intramuscularly. Further provided herein is a method in which the composition is administered intranasally. Further provided herein is a method in which the subject has, is suspected of having, or is at risk of developing an RSV infection. Further provided herein is a method in which the subject generates an immune response to an antigen. Further provided herein is a method in which the immune response comprises increasing the titer of neutralizing antibodies to the antigen compared to a subject not receiving the composition. Further provided herein is a method in which the immune response comprises increasing the amount of CD4+ and / or CD8+ positive T cells compared to a subject not receiving the composition. Further provided herein are methods, wherein the subject is immunocompromised or immunosuppressed.

[0130] Further provided herein is a kit comprising the composition provided herein, a package and materials therefor.Further provided herein is a kit comprising: (a) a lipid carrier that is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids;(b) at least one nucleic acid sequence that encodes a sequence capable of expressing an antigen, the antigen being derived from an infectious agent.Further provided herein is a kit comprising one or more surfactants.

[0131] 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 an infectious disease antigen. Further provided herein is a composition wherein the infectious disease antigen is derived from a virus that is influenza, VZV, RSV, human gammaherpesvirus 4, human papillomavirus (HPV), rabies virus, human alphaherpesvirus 1, or human alphaherpesvirus 2. Further provided herein is a composition wherein the infectious disease antigen is an influenza hemagglutinin protein stem region or a functional variant thereof. Further provided herein is a composition wherein the infectious disease antigen is derived from Mycobacterium, Streptococcus, Pseudomonas, Salmonella, Staphylococcus, Neisseria, Clostridium, or Chlamydia. Further provided herein is a composition wherein the first nucleic acid and the second nucleic acid are present on a common nucleic acid. Further provided herein is a composition wherein the first nucleic acid and the second nucleic acid 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.

[0132] The following examples are presented to more clearly illustrate to those skilled in the art the principles and practice of the embodiments disclosed herein, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are by weight. EXAMPLES

[0133] Example 1 Immunogenicity of RSV replicons To determine the biological activity of lipid carriers complexed with RSV repRNA and to measure the in vivo production of anti-RSV IgG, an assay was designed to first immunize 6-8 week old C57BL / 6 and BALB / c female mice with RSV repRNA and then analyze the levels of anti-RSV target IgG in the serum.

[0134] The formulation was prepared by combining repRNA encoding RSV-F and RSV-G proteins (approximately 10 ng / μL, −80° C.) with lipid carrier formulation (30 mg DOTAP / mL, 4° C.). 144 microliters (μL) of the formulation was then mixed with 180 μL of 40% sucrose and 36 μL of 100 mM citrate and divided into 2 mL tubes. The tubes were inverted 10 times to mix thoroughly.

[0135] Fifty microliters of the solution was injected intramuscularly into each mouse strain on day 0 to yield a 2.5 μg RNA dose, and blood was collected by retro-orbital eye bleed on days 14, 28, and 75 post-injection. Once collected, blood was allowed to clot and serum was collected and stored at -80 degrees Celsius until evaluation.

[0136] Blood draws were assessed by ELISA for IgG concentrations in response to RSV recombinant proteins. Three recombinant proteins were used (1 μg / mL): RSV-F protein; RSV-G protein with a C-terminal His tag from respiratory syncytial virus B1, a recombinant from RSV; and RSV-G protein from RSV purified from HEp-2 cells (A2). The data showed that RSV-F protein-specific responses were induced in both C57BL / 6 and BALB / c mice (Figure 2), while RSV-G A2 strain protein-specific responses were induced only in BALB / c mice (Figure 3A). No RSV-G B1 strain protein-specific responses were observed (Figure 3B). The lack of response to the B1 strain was not due to a failure of the ELISA reagents, since RSV-G A2 protein responses were detected on the same ELISA plate. Example 2 Lipid carrier innate immune response in macrophages

[0137] Various repRNA / lipid carrier formulations were prepared and analyzed to evaluate the innate immune response of lipid carriers in macrophages. The repRNA / lipid carrier formulations assayed included 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.

[0138] Protein expression and stimulation of tumor necrosis factor-alpha (TNFα) production by THP-1 macrophages were assayed. ELISA assays were performed to assess TNF-alpha (α) protein levels in culture media using Human TNF-alpha (α) DUOSET™ ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. 96-well microplates were coated with anti-TNFα capture antibody. The plates were blocked and culture media samples were added directly to the microplates without dilution. After addition of biotinylated detection antibody, streptavidin-horseradish peroxidase (SA-HRP), and substrate, absorbance was read at 450 nm in a SPECTRAMAX® i3 (Molecular Devices, LLC, San Jose CA, USA) plate reader.

[0139] First, THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA). Cells were then transfected with various formulations carrying replicon RNA or pathogen-associated molecular patterns (PAMPs)-encoding nanoluciferase (NanoLuc). Cell culture media was then evaluated for NanoLuc expression by luciferase assay and for TNFα expression by ELISA. The formulations used in the assays are listed in Table 3. [Table 3]

[0140] Eleven treatment groups were prepared. Eight of these groups were NanoLuc repRNA groups, and 600ng of repRNA dose per well was 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. Two of these groups were PAMP groups, and 200 nanogram (ng) dose per well was prepared using Fe-lipid carrier and Fe-lipid carrier miglyol formulations. The untreated group had neither NanoLuc repRNA nor PAMP.

[0141] Various formulations were prepared by diluting NanoLuc repRNA to 8 ng / μL in 2.2 mL of RNAse-free water and PAMP RNA to 2.67 ng / μL in 600 μL of water. The lipid carrier and RNA master mix were complexed by adding 250 μL of each diluted formulation to 250 μL of diluted RNA and mixed by pipetting up and down.

[0142] Cell transfections were performed at 7 × 10 cells per well in 24-well plates. 5The 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.

[0143] Protein expression levels from the first assay (FIG. 4A) and the second assay (FIG. 4B) were performed using a luciferase assay by first diluting the Nano-Glo® luciferase assay reagent 1:50 with buffer. 25 μL of supernatant was removed and mixed with 25 μL of Nano-Glo® reagent in a 96-well plate and incubated at room temperature for 3 minutes. Luminescence was read using a luminometer.

[0144] The studies described herein demonstrate that the miglyol lipid carrier formulation induced higher protein production from the replicon as shown in the first assay in Figure 4A and in the second assay in Figure 4B. A reduction in the innate immune response was detected as measured by TNF-α secretion as shown in Figures 5A-5B.

[0145] 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.

[0146] TNF-α protein levels in the medium from the first assay in FIG. 5A and the second assay in FIG. 5B were determined using a human TNF-α DUOSET™ ELISA (R&D Systems) according to the manufacturer's protocol. 96-well microplates were coated with anti-TNF capture antibody. The plates were 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. As shown in the first assay in FIG. 6A and in the second assay in FIG. 6B, a correlation between enhanced protein production and low TNF-α stimulation was observed with the miglyol lipid carrier formulation. The solanesol lipid carrier formulation induced slightly lower protein production but more TNFα production (FIGS. 6A-6B). The correlation data from the first assay is shown in Figure 6A and was derived from the data displayed in Figures 4A and 5 A. The correlation data from the second assay is shown in Figure 6B and was derived from the data displayed in Figures 4B and 5B. Example 3 NP-1 production and stability

[0147] Preparation of NP-1. NP-1 particles contain 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 Jose, CA, USA) and 10 mM sodium citrate dihydrate (Fisher Chemical). 1 ml of 20 mg Fe / ml 12 nm diameter oleic acid coated iron oxide nanoparticles (Imagion Biosystems, lot number 95-127) in chloroform was 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 left in a fume hood to completely evaporate, 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 the 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. 92 ml of the aqueous phase was transferred to another glass bottle and heated to 65°C 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 supplementary 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 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).

[0148] Preparation of NP-3. NP-3 particles contain 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). 1 ml of 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles (Imagion Biosystems, lot number 95-127) in chloroform was 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 left in a fume hood to completely evaporate, 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 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 another glass bottle and heated to 65° C. 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 supplementary 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 NP-1 was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8°C. Iron concentrations were determined by ICP-OES. DOTAP concentrations were measured by RP-HPLC.

[0149] Preparation of NP-30. A lipid carrier without inorganic core particles in the core was produced, having 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. 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 produce an oil phase. The oil phase was sonicated in a water bath at 65 degrees Celsius 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 to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 96 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 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 NP-30 without inorganic core 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.

[0150] Stability. Nanoparticles from NP-1 were placed in a stability chamber at the indicated temperatures. Stability was determined by particle size measurements using dynamic light scattering. Results show that the NP-1 formulation formed stable colloids when stored at 4, 25 and 42 degrees Celsius. Time measurements were performed over a 4 week period. The nanoparticle size range was approximately 50-100 nm in diameter, becoming closer to 40-60 nm in diameter over time for the 4 and 25 degree Celsius conditions. Example 4 Additional nanoparticle formulations Additional nanoparticle formulations are produced according to the following tables (Tables 4 and 5). The mRNA contains sequences encoding influenza virus antigens or VZV virus antigens (SEQ ID NOs: 3-5, and 38-43, 47). [Table 4] [Table 5] Example 5 Self-replicating mRNA construct 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 shown in SEQ ID NO:34. Example 6 Varicella-zoster virus (VZV) RNA vaccine composition

[0151] Six constructs were designed for a self-replicating (repRNA) vaccine against Varicella-Zoster Virus (VZV) encoding various combinations of glycoprotein E (gE) and glycoprotein I (gI). VZV gI was engineered to contain either a Thosea asigna virus 2A (T2A) ribosome skipping peptide or an internal ribosome entry site (IRES) to coordinate co-expression of downstream gE. Each self-replicating RNA construct is summarized in Table 6 and shown in Figure 7A. [Table 6]

[0152] Four micrograms (μg) of each corresponding RNA was transfected into baby hamster kidney epithelial cells (BHK) using Lipofectamine 2000 as a delivery vehicle. Cell lysates and cell supernatants were examined by Western blot and probed with anti-VZV-gE antibody at a 1:2000 dilution (Figure 7B). Supernatants from BHK transfected with secreted gE, VZV-FL-gE, and VZV-N-terminal trunc gE expressed anti-VZV-gE antibody.

[0153] Six-week-old female C57BL / 6 mice (n=5 mice per group) were immunized intramuscularly in the hind leg with 10 μg of repRNA encoding Varicella-Zoster Virus (VZV) antigens (Table 6, FIG. 7A). Serum was collected 14 days after vaccination. In addition, mice were immunized with a positive control containing repRNA encoding gE without the transmembrane sequence, thus resulting in only soluble gE. Serum antibody titers against soluble VZV gE were measured by ELISA after vaccination (FIG. 7C). Mice treated with RNA encoding an N-terminally truncated form of gE (VZV-N-terminal trunc gE) developed elevated antibody titers compared to control mice or those immunized with full-length gE. array

[0154] The sequences below (SEQ ID NOs: 38-43) are formatted to show the vector backbone and antigen open reading frames as follows: lower case letters indicate vector backbone sequences and upper case letters indicate VZV antigen open reading frames. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0155] The sequences below (SEQ ID NOs: 44-47) are formatted to show the vector backbone and antigen open reading frames as follows: lower case letters indicate VEEV replicon backbone sequences and upper case letters indicate antigen open reading frames. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0156] 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 by way of example only. Numerous variations, modifications, and substitutions will now 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 present disclosure described herein can be utilized in implementing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A composition comprising: A lipid carrier comprising: The lipid carrier is Liquid oil, cationic lipids, Hydrophilic surfactants, A lipid carrier comprising a hydrophobic surfactant, and At least one nucleic acid encoding a viral antigen sequence wherein the viral antigen sequence is derived from an influenza virus or a functional variant thereof. composition.

2. 2. The composition of claim 1, wherein the influenza virus is an influenza A virus or an influenza B virus.

3. The composition described in claim 1, wherein at least one of the nucleic acids comprises an antigenic sequence encoding an influenza hemagglutinin protein stem region or a functional variant thereof.

4. The composition of claim 1 , wherein the at least one nucleic acid comprises a sequence that is at least 85% identical to SEQ ID NO:

3.

5. The composition of claim 1, wherein the at least one nucleic acid comprises a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 10-12.

6. The composition of claim 1 , wherein the at least one nucleic acid is in a complex with the lipid carrier.

7. The composition of claim 1 , wherein the at least one nucleic acid further encodes an RNA polymerase.

8. 8. The composition of claim 7, wherein the RNA polymerase is Venezuelan equine encephalitis virus (VEEV) RNA polymerase.

9. The composition of claim 7 , wherein the at least one nucleic acid encoding the RNA polymerase comprises SEQ ID NO:

34.

10. 2. The composition of claim 1, 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, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E.

11. 11. The composition of claim 10, wherein the triglyceride is capric triglyceride, caprylic triglyceride, caprylic and capric triglycerides, triglyceride esters, or triglyceryl myristate.

12. The composition of claim 1 , wherein the lipid carrier comprises a hydrophobic core.

13. The composition of claim 1 , wherein the lipid carrier further comprises inorganic particles.

14. The composition of claim 13 , wherein the inorganic particles are within the hydrophobic core of the lipid carrier.

15. The composition of claim 13 , wherein the inorganic particles comprise a metal.

16. The composition of claim 15 , wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.

17. 17. The composition of claim 16, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.

18. The composition of claim 1 , wherein the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate.

19. The composition of claim 1 , wherein the hydrophilic surfactant is a polysorbate.

20. The cationic lipid is 1,2-dioleoyloxy-3(trimethylammonium)propane, 3β-[Ν-(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), bis(azanetriyl)-2-hexyl decanoate, 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 DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, 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))hexanonanoate, The composition of claim 1, comprising butadecan-9-yl 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.

21. The composition of claim 1 which is lyophilized.

22. A pharmaceutical composition comprising the composition of claim 1 and a pharma- ceutically acceptable excipient.

23. 13. Use of the 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 composition is administered to the subject.

24. The composition of claim 1 for generating an immune response in a subject, characterized in that a therapeutically effective amount of the composition is administered to the subject.

25. A lipid carrier comprising: The lipid carrier is Liquid oil, A surfactant, the surfactant comprising a hydrophilic surfactant and a hydrophobic surfactant; and a lipid carrier comprising a cationic lipid; Nucleic acid and wherein said nucleic acid comprises at least one nucleic acid encoding an antigenic sequence, said antigenic sequence comprising a sequence encoding a viral antigenic sequence, a bacterial antigenic sequence, a fungal antigenic sequence, a parasitic antigenic sequence, or a functional variant of any of the foregoing, and wherein said viral antigenic sequence is not a SARS-CoV-2 antigenic sequence or a functional variant thereof.

26. The composition of claim 25, wherein the viral antigen sequence is derived from varicella-zoster virus (VZV), respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), human gammaherpesvirus 4, human papillomavirus (HPV), rabies virus, human alphaherpesvirus 1, human immunodeficiency virus (HIV) or human alphaherpesvirus 2, or a functional variant of any of the foregoing.

27. The composition of claim 25, wherein the bacterial antigen sequence is derived from Mycobacterium, Streptococcus, Pseudomonas, Salmonella, Staphylococcus, Neisseria, Clostridium, or Chlamydia, or a functional variant of any of the foregoing.

28. The composition of claim 25, wherein the fungal antigen sequence is derived from an Aspergillus fungus, a Saccharomyces fungus, a Cryptococcus fungus, a Coccidioides fungus, a Neurospora fungus, a Histoplasma fungus, or a Blastomyces fungus, or a functional variant of any of the foregoing.

29. The composition of claim 25, wherein the nucleic acid comprises a sequence encoding an RNA polymerase.

30. The composition of claim 25 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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