RNA adjuvants, methods and uses thereof

Abstract

Provided herein are adjuvants, compositions, and methods for the prevention and treatment of infectious diseases and cancer. Provided herein are various toll-like receptor (TLR) agonists and RNAs encoding the TLR agonists. The adjuvants provided herein can be compounded with a carrier or formulated with a delivery vehicle for administration to a subject. Also provided are adjuvants that can be delivered with or as part of a vaccine composition to enhance an innate immune response in a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 347,635, filed on June 1, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence Listing

[0004] This application incorporates by reference a sequence listing XML submitted through the USPTO patent electronic application system. The sequence listing XML named 207481-701601_SL.xml was created on May 31, 2023 and is 427,686 bytes in size. Background Art

[0005] RNA vaccines have become a promising therapy for preventing the spread of infectious diseases such as COVID-19. However, RNA vaccines are expensive, require high doses of RNA per vaccine, and lack long-term efficacy in preventing infectious diseases. In addition, RNA is translated using a host cell mechanism regulated by the interferon system that can reduce the amount of RNA translated in vivo. In addition, current vaccine compositions can cause undesirable side effects. Therefore, there is a need for adjuvants that can reduce RNA vaccine side effects, optimize RNA dosage in RNA vaccines, and promote innate and adaptive immune responses that enhance the effectiveness of RNA vaccines. Summary of the invention

[0006] Provided herein are compositions for inducing an immune response upon administration to a subject, the compositions comprising: (a) a nucleic acid (e.g., RNA, DNA, or RNA adjuvant) encoding a synthetic protein, wherein the synthetic protein comprises at least one functional fragment of a toll-like receptor 5 (TLR5) agonist that is capable of activating a TLR5 pathway in a cell when in contact with a cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein upon administration of an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject.

[0007] Provided herein are compositions, wherein these compositions comprise: (a) a carrier (e.g., a lipid carrier); and (b) an RNA sequence encoding at least one functional fragment of a bacterial movement protein, wherein the functional fragment of the bacterial movement protein is a TLR5 agonist. In some embodiments, the functional fragment activates the TLR5 pathway in the cell when in contact with the cell.

[0008] Provided herein are compositions for inducing an immune response upon administration to a subject, the compositions comprising: (a) a nucleic acid (e.g., RNA, DNA, or RNA adjuvant) encoding a synthetic protein, wherein the synthetic protein comprises at least one functional fragment of a toll-like receptor 4 (TLR4) agonist that is capable of activating a TLR4 pathway in a cell when in contact with a cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein upon administration of an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject.

[0009] Provided herein are compositions, wherein the compositions comprise: (a) a carrier (e.g., a lipid carrier); and (b) one or more RNA sequences, wherein the one or more RNA sequences encode at least a functional fragment of a TLR4 agonist, wherein the functional fragment of the TLR4 agonist is capable of activating a TLR4 pathway in a cell when in contact with a cell.

[0010] Provided herein are compositions for inducing an immune response upon administration to a subject, the compositions comprising: (a) a nucleic acid (e.g., RNA, DNA, or RNA adjuvant) encoding a synthetic protein, wherein the synthetic protein comprises at least one functional fragment of a toll-like receptor 2 (TLR2) agonist that is capable of activating the TLR2 pathway in a cell when in contact with a cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein upon administration of an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject.

[0011] Provided herein are compositions, wherein the compositions comprise: (a) a carrier (e.g., a lipid carrier); and (b) one or more RNA sequences, wherein the one or more RNA sequences encode at least a functional fragment of a TLR2 agonist, wherein the functional fragment activates a TLR2 pathway in a cell when in contact with the cell.

[0012] Provided herein are compositions for inducing an immune response after administration to a subject, the compositions comprising: (a) one or more carriers (e.g., lipid carriers); (b) one or more nucleic acids (e.g., RNA or DNA), wherein the one or more nucleic acids encode: (i) at least one functional fragment of a TLR4 agonist, a TLR2 agonist, or a TLR5 agonist, or any combination thereof; (ii) at least one RNA encoding a viral antigen, or (iii) a combination of (i) and (ii); and (c) a delivery vehicle, wherein after administration of an effective amount of the composition, an innate immune response is induced in the subject in the presence of a viral antigen, thereby inducing an immune response to the viral antigen. In some embodiments, the delivery vehicle is formulated for intranasal administration, oral administration, intradermal administration, intranodal administration, intrasplenic administration, intratumoral administration, intravenous administration, or intramuscular administration.

[0013] Provided herein are compositions comprising: (a) an RNA adjuvant comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2 to 175; and (b) a delivery vehicle, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0014] Provided herein are compositions comprising: (a) a DNA sequence encoding an RNA adjuvant comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2 to 175; and (b) a delivery vehicle, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0015] Compositions are provided herein, wherein the compositions comprise: (a) a nucleic acid encoding a synthetic protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 176 to 217; and (b) a delivery vehicle, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof. Also provided herein are nucleic acids encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 206. Also provided herein are nucleic acids encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 210. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 212. Also provided herein are nucleic acids encoding an amino acid sequence having at least 85% identity to SEQ ID NO: 213. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 215. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 217.

[0016] Provided herein are compositions comprising: (a) a synthetic protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 176 to 217; and (b) a delivery vehicle, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0017] Provided herein are compositions, wherein the compositions comprise: a synthetic protein comprising at least one protein construct, wherein at least one protein construct comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 176 to 217. Also provided herein are compositions comprising two or more, three or more, four or more, or five or more protein constructs.

[0018] Provided herein are compositions, wherein the compositions comprise: nucleic acids encoding synthetic proteins comprising at least one protein construct, wherein at least one protein construct comprises an amino acid sequence selected from SEQ ID NOs: 176 to 217. Also provided herein are compositions comprising two or more, three or more, four or more, or five or more protein constructs.

[0019] Provided herein are compositions, wherein the compositions comprise: (a) yeast cells, wherein the yeast cells comprise a permeable cell wall; and (b) one or more nucleic acids (e.g., DNA, RNA, or a combination thereof), wherein the one or more nucleic acids encode at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof, wherein the functional fragment activates a TLR4, TLR5, or TLR2 pathway in the cell when in contact with the cell.

[0020] Provided herein are compositions for inducing an immune response after administration to a subject, wherein these compositions comprise: (a) one or more carriers (e.g., lipid carriers); (b) one or more nucleic acids, wherein one or more nucleic acids encode: (i) at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or any combination thereof; (ii) a tumor antigen; (iii) an immunomodulatory protein; or (iv) a combination thereof; and (c) a delivery vehicle, wherein after administration of an effective amount of the composition, an innate immune response is induced in the subject in the presence of a tumor antigen, thereby inducing an immune response against the tumor antigen. In some embodiments, the functional fragment activates TLR4, TLR5, or TLR2 pathways in the cell when in contact with the cell.

[0021] Provided herein are vectors, wherein the vectors comprise: one or more nucleic acids (eg, DNA, RNA, or a combination thereof) encoding: a TLR5 agonist, a TLR4 agonist, a TLR2 agonist, or a combination thereof.

[0022] Provided herein are compositions, wherein the compositions comprise a vector provided herein; and a carrier (eg, a lipid carrier).

[0023] Provided herein are vaccine compositions, wherein the vaccine compositions comprise a composition provided herein; and a pharmaceutically acceptable excipient.

[0024] Provided herein are vaccine compositions, wherein the vaccine compositions comprise: (a) a lipid carrier; (b) one or more RNA adjuvants, wherein the one or more RNA adjuvants comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 175; and (c) one or more RNA sequences encoding antigens. Also provided herein are vaccine compositions, wherein the one or more RNA sequences encoding antigens each encode a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or any combination thereof.

[0025] Provided herein are vaccine compositions, wherein these vaccine compositions comprise: (a) a lipid carrier; (b) one or more nucleic acids encoding RNA adjuvants provided herein, wherein the one or more RNA adjuvants comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 175; and (c) one or more nucleic acids encoding antigens. Also provided herein are vaccine compositions, wherein the one or more nucleic acids encoding antigens each encode a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, a tumor antigen, or any combination thereof. In some embodiments, the one or more nucleic acids comprise DNA. In some embodiments, the one or more nucleic acids comprise RNA. In some embodiments, the one or more nucleic acids encoding RNA adjuvants are DNA, RNA, or a combination thereof.

[0026] Provided herein is a method for stimulating an immune response in a subject, the method comprising: administering to a subject an effective amount of a composition provided herein, a vector provided herein, or a vaccine composition provided herein, thereby stimulating an immune response. Provided herein is a method for enhancing an immune response to an antigen encoded by a nucleic acid (e.g., RNA) in a vaccine composition (e.g., RNA vaccine), the method comprising: (a) administering to a subject an effective amount of a composition provided herein or a vaccine composition provided herein; and (b) administering to a subject an RNA vaccine composition, wherein the RNA vaccine composition comprises RNA encoding an antigen, thereby enhancing an immune response to an antigen encoded by RNA. Provided herein is a method for treating an infection in a subject, the method comprising: administering to a subject an effective amount of a composition provided herein, a vector provided herein, or a vaccine composition provided herein, thereby treating an infection in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments that illustrate the principles of the present invention and the accompanying drawings, in which:

[0028] Figure 1 is a schematic diagram providing the mechanism of action of the RNA adjuvants provided herein.

[0029] FIG. 2A to FIG. 2D is a schematic diagram showing several configurations of RNA adjuvants and RNA sequences encoding antigens. Figure 2A A schematic diagram of Configuration 1 is shown. Figure 2B A schematic diagram of Configuration 2 is shown. Figure 2C A schematic diagram of Configuration 3 is shown. Figure 2D A schematic diagram of Configuration 4 is shown.

[0030] FIG. 3A to FIG. 3C Shown are schematic diagrams and graphs demonstrating the effects of RNA-encoded TLR5 agonists (SEQ ID NO: 2, SEQ ID NO: 176) on HEK293 cells with a nuclear factor-κB (NF-κB) luciferase reporter gene and HEK293 cells without the reporter gene. Figure 3A Schematic diagram of the experimental protocol for cell transfection and cell conditions is shown. Figure 3B A graph showing the change in luciferase activity (RLU) driven by NF-κB activation over time (0 hours, 18 hours and 40 hours after transfection) is shown. Y-axis: luciferase activity; X-axis: hours after transfection for each condition indicated. Figure 3CA graph showing the change in TLR5 agonist concentration over time (0 hours, 18 hours and 40 hours after transfection) is shown. Y-axis: TLR5 agonist concentration (ng / mL); X-axis: hours after transfection for each condition indicated.

[0031] FIG. 4A to FIG. 4B Graph showing the effect of lipid nanoparticle (LNP) encapsulated toll-like receptor agonist RNA on packaging and NF-κB signaling. Figure 4A A schematic diagram of the experimental protocol and a graph showing the luciferase activity of HEK293 cells treated with TLR5 agonist 1 RNA (LNP) encapsulated in lipid nanoparticles, TLR5 agonist protein alone, or control LNP (GFP) over time. Luciferase activity driven by NF-κB activation was measured at different time points 48 hours after transfection. Y-axis: luciferase activity (RLU); X-axis: hours after treatment with LNP (TLR5 agonist 1 RNA), TLR5 agonist 1 protein, or LNP (GFP). Figure 4B A schematic diagram of the experimental scheme and a graph of the luciferase activity of secreted TLR5 agonist proteins from cell culture medium and cell culture relative to LNPs without RNA encoding TLR5 agonists are shown. Y axis (left): luciferase activity (RLU); Y axis (right): fluorescence signal (RFU); X axis: hours after treatment with TLR5 agonist 1 protein or LNPs (TLR5 agonist 1 RNA).

[0032] FIG. 5A to FIG. 5B Schematic diagram and graph showing the effect of TLR5 agonist 1 on HEK293-hTLR5-Luc and HEK293-null-Luc cells. Figure 5A A schematic diagram of the experimental scheme and a graph of TLR5 agonist protein release after LNP+TLR5 agonist transduction into HEK293 cells with a human TLR5-luciferase reporter gene relative to HEK293 cells that do not express TLR5 are shown. Cells were sampled up to 23 hours after LNP+TLR5 agonist delivery. Y-axis: indicated conditions; X-axis: luciferase activity (RLU). Figure 5B Graph showing TLR5 agonist 1 protein release after transduction of LNPs (TLR5 agonist 1) into HEK293-hTLR5-Luc and HEK293-null-Luc cells. Y-axis: TLR5 agonist protein (ng / mL); X-axis: hours after treatment.

[0033] Figure 6Luminescence images showing the effect of LNP (mRNA TLR5 agonist) in BALB / c-Tg (IκBα-luc) Xen NF-κB luciferase reporter mice. Mice were administered LNPs encapsulating mRNA encoding TLR5 agonist 1, PBS, or LNPs with GFP reported alone. At 5 hours after treatment, mice treated with LNP (TLR5 agonist 1 RNA) had the highest level of luminescence relative to other conditions.

[0034] Figure 7 A graph showing the kinetics of the response to LNP (mRNA encoding TLR5 agonist 1) in vivo (NF-κB reporter mouse) at 0, 3, 5, 6, 8, 10, 12, 15, 18, and 36 hours after injection is shown. From left to right, the bars show LNP (TLR5 agonist 1); LNP (GFP); and PBS control. Y axis: total luminescence; X axis: hours after injection.

[0035] Various aspects will now be described more fully below. However, these aspects can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein; on the contrary, these embodiments are provided to make this disclosure thorough and complete and to fully convey its scope to those skilled in the art. DETAILED DESCRIPTION

[0036] Provided herein are RNA adjuvant compositions and vaccine compositions, methods, and uses thereof for inducing immune responses against antigens to prevent infectious diseases and treat cancer. In short, further described herein are (1) RNA adjuvant compositions; (2) nucleic acids encoding antigens; (3) lipid carriers; (4) yeast cell compositions; (5) pharmaceutical vaccine compositions, dosages, and administration; and (6) therapeutic applications.

[0037] definition

[0038] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0039] All references, patents and patent applications disclosed herein are incorporated herein by reference with respect to each cited subject matter, and in some cases, they may encompass the entire document. All references disclosed herein (including patent references and non-patent references) are incorporated herein by reference in their entirety, just as if each were individually incorporated. However, in the case where patents, patent applications or publications containing clear definitions are introduced by reference, those clear definitions should be understood to apply to the incorporated patents, patent applications or publications in which they are found, and not necessarily to the text of the present application, particularly the claims of the present application, in which case the definitions provided herein are meant to replace.

[0040] The indefinite article "a / an" used in the specification and claims should be understood to mean "at least one" unless explicitly stated to the contrary.

[0041] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately 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 combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether or not related to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to only A (optionally including elements other than B) in one embodiment; to only B (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; and so on.

[0042] As used 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 inclusive, i.e., including at least one, but also including more than one multiple or a series of elements, and optionally, additional unlisted items. Only terms that clearly indicate the opposite, such as "only one" or "exactly one", or when used in the claims, "consisting of..." will refer to including exactly one element in a plurality or a series of elements. In general, the term "or" as used herein should only be interpreted as indicating an exclusive choice (i.e., "one or the other, but not both") when preceded by an exclusive term, such as "either", "one", "only one" or "exactly one". When used in the claims, "essentially consisting of..." should have the ordinary meaning used in the field of patent law.

[0043] As used herein, "optional" or "optionally" means that the subsequently described event may or may not occur, and therefore, the description includes instances where the event occurs and instances where it does not.

[0044] As used herein, the term "about" or "approximately" refers to a range of up to ±20% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. When specific values ​​are described in the application and claims, unless otherwise indicated, the term "about" is implicit and means in this context that it should be assumed to be within an acceptable error range for the specific value.

[0045] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect.

[0046] (1) RNA adjuvant composition

[0047] Compositions comprising nucleic acids are provided herein. Compositions comprising RNA adjuvants are also provided herein. Compositions (e.g., pharmaceutical compositions and vaccine compositions) are also provided herein, wherein the vaccine composition comprises nucleic acids (e.g., RNA adjuvants provided herein) and nucleic acids encoding antigens (e.g., RNA). In some embodiments, nucleic acids encoding activation toll-like receptors (TLRs) are provided herein. In some embodiments, nucleic acid activation TLRs. Based on their location, TLRs are mainly divided into two subfamilies: cell surface TLRs and intracellular TLRs. Cell surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, while intracellular TLRs are located in endosomes and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13. Cell surface TLRs typically recognize microbial membrane components, such as lipids, lipoproteins, and proteins. For example, TLR4 recognizes bacterial lipopolysaccharides (LPS). TLR2 and TLR1 or TLR6 together recognize a variety of PAMPs, including lipoproteins, peptidoglycans, lipoteichoic acid, zymosan, mannan, and tGPI-mucin. TLR5 recognizes bacterial flagellin. Human TLR10 cooperates with TLR2 to recognize ligands from Listeria. TLR10 can also sense influenza A virus infection.

[0048] TLRs recognize pathogen-associated molecular patterns (PAMPs) derived from microbial organisms and signal by recruiting specific adapter molecules, leading to the activation of transcription factors (e.g., NF-κB), thereby regulating the innate immune response to PAMPs or microbial organisms expressing PAMPs. The innate immune response is the first line of defense against invading pathogens. The innate immune response is a non-antigen-dependent or non-specific defense mechanism used by the host immediately or within hours after encountering an antigen. The innate immune response has no immunological memory, and therefore, if the body is exposed to the same pathogen in the future, it cannot recognize or "remember" the pathogen. On the other hand, adaptive immunity is antigen-dependent and antigen-specific, and therefore, there is a lag time between exposure to an antigen and the maximum response. The hallmark of adaptive immunity is the ability to remember, which enables the host to produce a more rapid and effective immune response when subsequently exposed to an antigen. Innate immunity and adaptive immunity are not mutually exclusive host defense mechanisms, but are complementary, and defects in any one system can lead to vulnerability or inappropriate responses in the host.

[0049] The advantage of the composition provided herein is that RNA adjuvants enhance the innate immune signaling pathway through TLR signaling while promoting adaptive immune responses. Compared with vaccine compositions that do not contain mRNA adjuvants, this effect can reduce the effective amount of RNA required for encoding antigens in the vaccine composition, thereby reducing the dose required for mRNA encoding antigens. The mRNA adjuvant provided herein activates nuclear factor-κB (NF-κB) signaling downstream of TLRs necessary for T cell activation. Unlike other TLRs, the activation of NF-κB mediated by TLR5 rarely involves the production of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), because the unique tissue specificity of TLR5 expression is mainly limited to epithelial and endothelial cells, and does not involve T cells and macrophages. This contributes to the safety of mRNA vaccines combined with adjuvants based on TLR5 agonists, because TNF-α and IL-1β can cause negative side effects, such as septic shock, fever and application site irritation. In addition, the mRNA adjuvant provided herein attracts and promotes the adaptive immune response of the immune system targeting antigens and the long-term memory of pathogens.

[0050] The nucleic acid (e.g., RNA adjuvant) provided herein encodes a TLR agonist or a functional fragment thereof. The functional fragment is capable of activating a TLR pathway or TLR in a cell when in contact with a cell. In some embodiments, a TLR agonist directly binds to a TLR. In some embodiments, a TLR agonist or a functional fragment thereof is a naturally occurring TLR agonist, protein, or nucleic acid. In some embodiments, a TLR agonist or a functional fragment thereof is a non-naturally occurring TLR agonist, protein, or nucleic acid. In some embodiments, a non-naturally occurring TLR agonist is an RNA sequence encoding a TLR agonist protein. In some embodiments, a TLR agonist or a functional fragment thereof is a synthetic protein (also referred to as an engineered protein). The synthetic protein provided herein is non-naturally occurring and can be engineered using known methods, such as molecular cloning techniques. In some embodiments, the compositions provided herein include a variety of mRNA adjuvants provided herein. In some embodiments, the mRNA adjuvant provided herein activates TLR2; TLR4; and / or TLR5.

[0051] In some embodiments, activation of a TLR provided herein increases the level or activity of NF-κB. NF-κB is a transcription factor that regulates the response of cells to infectious agents and serves as a mediator of innate and adaptive immune responses. NF-κB also mediates the production of proinflammatory cytokines.

[0052] In some embodiments, the compositions provided herein include at least one functional fragment of a TLR5 agonist, wherein the TLR agonist includes a functional fragment of a bacterial movement protein. In some embodiments, the bacterial movement protein is a bacterial flagellin, a derivative thereof or a functional fragment. In some embodiments, the bacterial movement protein provided herein is a truncated form of a bacterial flagellin, a secreted form of a bacterial flagellin or a combination thereof. Flagellin includes two to four domains. Common D0 and D1 domains are hidden in the core of the flagellar filament by mediating the interaction between flagellins, and are conserved in bacterial species due to their functional importance in filament formation. The flagellin D1 domain has a common molecular pattern of TLR5 interactions, although the sequence and domain between flagellins are different. D0 and D1 domains stimulate TLR5 activation. In three domains and four domain flagellins, the D1 domain extends to the auxiliary domains (D2 and D3) located on the surface of the flagellar filament. In contrast to D0 and D1, D2 or D3 domains show significant differences in sequence and structure and are considered to activate adaptive immunity. Many Gram-positive bacteria (such as Bacillus subtilis and Clostridium difficile) express flagellin lacking hypervariable domains, and therefore contain the minimum region (D0 domain and D1 domain) required for TLR5 activation and flagellin polymerization into flagellar filaments. Surprisingly, not all bacterial flagellins can activate TLR5. For example, flagellin from Campylobacter jejuni and Helicobacter pylori belonging to the ε-Proteobacteria does not induce TLR5 signal transduction, and can escape TLR5-mediated immune surveillance. The main functional differences between TLR5-activated flagellin and non-activating factors can be attributed to sequence variation.

[0053] The proteins provided herein (e.g., synthetic proteins or proteins encoded by nucleic acids provided herein) may comprise two (D0 and D1), three (D0, D1 and D2) or four (D0, D1, D2 and D3) domains from bacterial flagellin. Compositions are provided herein, wherein the functional fragments of toll-like receptor 5 (TLR5) agonists comprise the D0 region and the D1 region of bacterial flagellin. In some embodiments, the functional fragments of toll-like receptor 5 (TLR5) agonists comprise the D0 region, the D1 region and the D2 region of bacterial flagellin. In some embodiments, the functional fragments of toll-like receptor 5 (TLR5) agonists comprise the D0 region, the D1 region, the D2 region and the D3 region of bacterial flagellin.

[0054] In some embodiments, the compositions provided herein include at least one functional fragment of a TLR4 agonist. In some embodiments, the TLR4 agonist includes a high mobility group protein B1 (HMGB1) protein, nicotinic acid ester-nucleotide-dimethylbenzimidazole phosphoribosyl transferase, transglycosylase, 50S ribosomal protein, heparin binding hemagglutinin protein, YadA family autotransport adhesin, dnaJ protein, pneumococcal hemolysin protein, Omp19 protein, 6,7-dimethyl-8-ribosylcytosine synthase or at least one functional fragment of any combination thereof. In some embodiments, the functional fragment activates the TLR4 pathway in the cell when in contact with the cell.

[0055] In some embodiments, the compositions provided herein include at least one functional fragment of a TLR2 agonist. In some embodiments, the TLR2 agonist includes at least one functional fragment of an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system. In some embodiments, the functional fragment activates the TLR2 pathway in the cell when in contact with the cell.

[0056] Proteins encoded by nucleic acids provided herein (e.g., DNA sequences, RNA sequences, or RNA adjuvants) can be designed by amino acid sequences of any bacteria or mammalian species encoding immunostimulatory pathways. Synthetic proteins provided herein can be chimeric proteins comprising protein fragments derived from two or more different organisms to form functional TLR agonists of activated TLR receptors, such as human TLR5, human TLR4, or human TLR2.

[0057] The protein encoded by the RNA adjuvant or nucleic acid provided herein may include modifications that improve the stability, targeting or function of the protein. In some embodiments, the protein includes a signal peptide for targeted cell localization. In some embodiments, the protein provided herein also includes the following signal sequence: MRSLSVLALLLLLLLAPASA (SEQ ID NO: 1). In some embodiments, the protein provided herein does not include a signal sequence. In some embodiments, the protein provided herein also includes an N-terminal spoke region. In some embodiments, the N-terminal spoke region includes the following sequence: SGLRINSAKDDA (SEQ ID NO: 218). In some embodiments, the protein provided herein also includes a joint. A joint is a molecular entity that can directly or indirectly connect two parts of a composition, such as a first protein construct and a second protein construct. The joint can be configured according to specific needs, such as stability or length between the first protein construct and the second protein construct. As another example, the joint can be configured to have sufficient length and flexibility. In some embodiments, the joint can be configured to promote expression and purification of the protein provided herein. In some embodiments, the joint includes the following sequence: SPG. In some embodiments, the protein includes a C-terminal spoke region. In some embodiments, the C-terminal spoke region comprises the following sequence: EDADYA (SEQ ID NO: 220). In some embodiments, the protein provided herein comprises a thrombin cleavage site. In some embodiments, the thrombin cleavage site comprises the following sequence: LVPRGS (SEQ ID NO: 221). In some embodiments, the protein provided herein comprises a peptide tag. In some embodiments, the peptide tag is a histidine (His) tag (e.g., comprising the following sequence: HHHHHH (SEQ ID NO: 222)).

[0058] The compositions provided herein may include any one of the RNA adjuvants or the sequences listed in Table 1, their derivatives, variants or functional fragments. The compositions provided herein may also include a DNA sequence encoding an RNA adjuvant or an amino acid sequence listed in Table 1, its derivatives, variants or functional fragments. In some embodiments, the RNA adjuvants provided herein encode synthetic proteins and / or TLR agonists, their derivatives or functional fragments, which contain at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence similarity with the sequences listed in Table 1. In some embodiments, the nucleic acid sequences or amino acid sequences provided herein contain sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with the sequences listed in Table 1. In some embodiments, the RNA sequences provided herein contain the same sequences as those listed in Table 1, or encode the same amino acid sequences as those listed in Table 1. The percentage (%) of sequence identity (or similarity) of a given sequence relative to a reference sequence is defined as the percentage of identical residues identified after comparing the two sequences and introducing a gap when necessary to obtain the maximum percentage of sequence identity. Percent identity can be calculated using alignment methods known in the art, for example, publicly available software such as BLAST, Align, ClustalW2 can be used to perform sequence alignment. Suitable alignment parameters can be determined by those skilled in the art, but the default parameters of BLAST are particularly contemplated.

[0059] In some embodiments, the compositions provided herein include the sequences listed in Table 1; and further include one or more sequence modifications. In some embodiments, the sequences provided herein are codon-optimized sequences. Sequence modifications may include, for example, substitution, deletion, insertion, chemical modification of one or more nucleobases; or chemical modification of the phosphate backbone, nucleotides, nucleobases, or nucleosides. Such modifications may be made to RNA adjuvant sequences, DNA sequences encoding RNA adjuvant sequences provided herein, or any sequence disclosed herein (e.g., mRNA encoding antigens). Methods for modifying nucleic acid or amino acid sequences are known. One of ordinary skill in the art will appreciate that modifications may be located at any position of the nucleic acid so that the function of the nucleic acid or synthetic protein is not significantly reduced. For example, software may be used to match identical or similar sequences by assigning homology to various substitutions, deletions, and / or other modifications. Nucleic acids provided herein may be prepared according to any available technique, including but not limited to chemical synthesis, enzymatic synthesis (commonly referred to as in vitro transcription), cloning, enzymatic or chemical cutting, etc. In some cases, nucleic acids provided herein are not uniformly modified along the full length of the molecule. Different nucleotide modifications and / or backbone structures may be present at different positions within the nucleic acid.

[0060] Table 1. RNA adjuvants.

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence having at least 90% identity to any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence having at least 95% identity to any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence selected from any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise SEQ ID NO: 2, a variant or a functional fragment thereof. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise SEQ ID NO: 62, a variant or a functional fragment thereof. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise SEQ ID NO: 98, a variant or a functional fragment thereof.

[0073] In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) encode an amino acid sequence having at least 75% identity to a sequence selected from any one of SEQ ID NOs: 176 to 204. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants or DNA) encode any one of SEQ ID NOs: 176, SEQ ID NOs: 181, SEQ ID NOs: 200, SEQ ID NOs: 201, SEQ ID NOs: 202, variants thereof, or functional fragments thereof. In some embodiments, the compositions provided herein comprise a protein comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 176 to 204. In some embodiments, the compositions provided herein comprise a protein comprising an amino acid sequence at least 85% identical to any one of SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202. In some embodiments, the compositions provided herein comprise a nucleic acid encoding an amino acid sequence at least 85% identical to any one of SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202.

[0074] Also provided herein is a composition comprising at least one protein construct. In some embodiments, the composition comprises two or more protein constructs (e.g., dimers). In some embodiments, the composition comprises three or more protein constructs (e.g., trimers). In some embodiments, the composition further comprises a joint between each protein construct. In some embodiments, the composition comprises one or more protein constructs selected from Table 2.

[0075] Table 2. Protein constructs.

[0076]

[0077]

[0078] In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 75% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 80% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 85% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 90% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 95% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 96% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 97% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 98% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence comprising at least 99% sequence identity with a sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acids (e.g., DNA, RNA, or a combination thereof) provided herein encode an amino acid sequence selected from any one of SEQ ID NOs: 205 to 217. In some embodiments, the nucleic acid provided herein (e.g., DNA, RNA or a combination thereof) encodes a leader peptide sequence. In some embodiments, the leader peptide sequence comprises the following amino acid sequence: MRSLSVLALLLLLLLAPASA (SEQ ID NO: 223). In some embodiments, the nucleic acid provided herein (e.g., DNA, RNA or a combination thereof) further encodes a zipper.For example, the zipper of the trimer peptide can comprise the following amino acid sequence: RMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO: 224), and the zipper of the dimer can comprise the following amino acid sequence: RMKQLEDKIEELLSKIYHLENEIARLKKLIGER (SEQ ID NO: 225).

[0079] Provided herein are synthetic proteins comprising at least one protein construct or a nucleic acid encoding at least one protein construct, wherein at least one protein construct comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a sequence selected from SEQ ID NOs: 176 to 217. In some embodiments, the synthetic proteins provided herein comprise sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NOs: 206, SEQ ID NOs: 210, SEQ ID NOs: 212, SEQ ID NOs: 213, SEQ ID NOs: 215, SEQ ID NOs: 217, or variants thereof. Also provided herein are compositions comprising two or more, three or more, four or more, or five or more protein constructs. In some embodiments, the nucleic acid encoding at least one protein construct is DNA. In some embodiments, the nucleic acid encoding at least one protein construct is RNA.In some embodiments, the nucleic acid comprises a self-replicating RNA sequence or a DNA encoding a self-replicating RNA sequence.

[0080] (2) Nucleic acid encoding antigen

[0081] Provided herein are compositions comprising one or more nucleic acids. In some embodiments, the one or more nucleic acids include deoxyribonucleic acid (DNA). In some embodiments, the one or more nucleic acids include ribonucleic acid (RNA). In some embodiments, the one or more nucleic acids include a mixture of DNA and RNA.

[0082] In some embodiments, the nucleic acid provided herein comprises an RNA adjuvant sequence (e.g., encoding a TLR agonist protein) and an antigen provided herein. In some embodiments, the nucleic acid provided herein comprises a polynucleotide comprising one or more modified nucleotides or core bases, and / or their analogs. The polynucleotide provided herein may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure may be modified before or after the composition provided herein is assembled. Modified nucleobases that can be incorporated into modified nucleosides and nucleotides and present in RNA molecules include: 1-methyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2-methyladenosine, 2-O-ribophosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-)- ... adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine, N6,2-O-dimethyladenosine, 2-O-methyladenosine, N6,N6,O-2-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5- Formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4,N4,2-O-trimethylcytidine, 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2-O-ribosylphosphate guanosine, 7-methylguanosine, undermodified hydroxyxanthine guanosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, 4-demethylguanosine, epoxyqueuosine, hydroxy wybutosine, isowyosine, N2,7,2-O-trimethylguanosine, N2,2-O-dimethylguanosine, 1,2-O-dimethylguanosine, 2-O-methylguanosine, N2,N2,2-O-trimethylguanosine, N2,N2,7-trimethylguanosine, peroxywybutosine, galactosyl-wybutosine, mannosyl-wybutosine, queuosine, archaeosine, wybutosine, methyl-glucosine, glucosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methyl 5-(carboxyhydroxymethyl)uridine, 5-(carboxymethylaminomethyl)uridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinemethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine Uridine, 3,2-O-dimethyluridine, 5-carboxymethylaminomethyl-2-O-methyluridine, 5-carbamoylmethyl-2-O-methyluridine, 5-methoxycarbonylmethyl-2-O-methyluridine, 5-(isopentenylaminomethyl)-2-O-methyluridine, 5,2-O-dimethyluridine, 2-O-methyluridine, 2-thio-2-O-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinemethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2-O-methylpseudouridine, inosine, 1-methylinosine, 1,2-O-dimethylinosine and 2-O-methylinosine. In some embodiments, the nucleic acids provided herein include pseudouridine modifications to ensure the stability of the mRNA. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the nucleic acid may contain aminophosphorothioate, phosphorothioate and / or methylphosphonate bonds. The RNA sequence or DNA sequence encoding the RNA adjuvant may be modified in terms of its codon usage, for example, to increase the translation efficiency and half-life of the RNA. A poly A tail (e.g., about 30 or more adenosine residues) may be attached to the 3' end of the RNA to increase its half-life. The cap structure may provide stability and translation efficiency for the RNA molecule. In some embodiments, the RNA adjuvant or DNA encoding the RNA adjuvant provided herein is chemically modified. In some embodiments, the nucleic acid encoding the antigen is chemically modified. In some embodiments, the nucleic acid encoding the antigen comprises DNA. In some embodiments, the nucleic acid encoding the antigen comprises RNA. In some embodiments, the chemical modification of the RNA sequence provided herein comprises a poly A tail, a chemically modified nucleobase provided herein, or a 5' end cap. ,

[0083] In some embodiments, RNA provided herein is mRNA. In some embodiments, mRNA is non-replicating mRNA. In some embodiments, mRNA is self-amplifying mRNA. Non-replicating mRNA and self-amplifying mRNA can utilize the translation mechanism of host cells to produce antigen targets and initiate adaptive immune response. Non-replicating mRNA encodes protein antigens of interest, while self-amplifying mRNA can also encode proteins, thereby allowing RNA replication.

[0084] Compositions are provided herein, wherein the composition comprises RNA encoding an antigen or multiple RNAs encoding different antigens. The compositions provided herein can be multivalent and used as vaccines to induce immune responses against multiple antigens (e.g., viruses, bacteria and / or tumor antigens). Compositions comprising RNA adjuvants provided herein and RNA encoding antigens are also provided herein. In some embodiments, the RNA sequence encoding the antigen is operably linked to the RNA adjuvant. Compositions comprising RNA adjuvants provided herein and protein antigens or fragments thereof are also provided herein. Protein antigens can include fragments of inactivated bacteria or viruses, bacterial toxins, or fragments of antigens expressed by microorganisms.

[0085] In some embodiments, the antigens provided herein are from a microorganism. In some embodiments, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen.

[0086] In some embodiments, the antigens provided herein are bacterial antigens. Non-limiting examples of infectious bacteria include: Escherichia coli (E. coli), Pseudomonas aeruginosa, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophilia, Mycobacterium (such as Mycobacterium tuberculosis, Mycobacterium avium (M. avium), Mycobacterium intracellulare (M. intracellulare), Mycobacterium kansasii (M. kansaii), Mycobacterium gordonii (M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Viridans group), Streptococcus faecalis, Streptococcus epidermidis, Streptococcus bovis, Streptococcus (anaerobic), Streptococcus pneumoniae, pathogenic Campylobacter, Enterococcus, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Brucella abortus, Pasteurella multocida, Bacteroides, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Nocardia brasiliensis, Borrelia hermsii, Borrelia burgdorferi, and Actinomyces israelii.

[0087] In some embodiments, the antigen provided herein is a viral antigen. In some embodiments, the antigen includes a spike protein, a glycoprotein, a hemagglutinin protein, or a viral envelope protein. In some embodiments, the viral antigen is from adenovirus, astrovirus, bocavirus, Banna virus, BK virus, Colti virus, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barr virus, Hanta virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus (HIV), influenza virus, JC virus, Lassa virus, Marburg virus, In some embodiments, the SARS virus is a SARS-CoV-2 virus.Other non-limiting examples of viruses include Retroviridae (e.g., HIV); Picornaviridae (e.g., poliovirus, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); Caliciviridae (such as strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus, West Nile virus, Zika virus); virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantaan virus, viruses, bunyaviruses, phleboviruses, and Nairoviruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (Adenoviridae). e) (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (such as African swine fever virus); cytomegalovirus (CMV) pneumonia, enteritis, and retinitis; Epstein-Barr virus (EBV) lymphoproliferative disease; varicella zoster virus (VZV); HSV-1 and HSV-2 mucositis; HSV-6 encephalitis, BK-virus hemorrhagic cystitis; and hepatitis A, B, or C.

[0088] Exemplary viral antigens that can be encoded by the RNA molecules provided herein include, but are not limited to, spike (S) protein, nucleocapsid protein (NP), hemagglutinin (HA), neuraminidase (NA), Den1 viral envelope protein, Den2 viral envelope protein, Den3 viral envelope protein, Den4 viral envelope protein, lipopeptides, gene products of HIV gag, pol and env genes, Nef protein, reverse transcriptase, nucleoprotein, matrix protein (M1), membrane protein (M2), transcriptase components (PB1, PB2 and PA), parvovirus antigens (e.g., VP-1, VP-2, VP-3, NS-1 and NS-2), RSV-F, RSV-G, rubella virus antigens (e.g., proteins E1 and E2), glycoprotein B, HPV antigens (e.g., HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35), Epstein-Barr nuclear antigen (EBNA)-1, E BNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA, EBV-VCA, HTLV-antigens (e.g., TAX), hepatitis B core antigen and hepatitis B envelope antigen, herpes simplex 1 / 2 (HSV1 / 2) HSV-2 glycoproteins B, C, D, E (gB2, gC2, gD2, gE2), cytomegalovirus (CMV) glycoprotein B (gB) and pentameric complex (PC).

[0089] In some embodiments, provided herein is an antigen of a fungus. Non-limiting examples of fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Pneumocystis carinii, Chlamydia trachomatis, and Candida albicans. In some embodiments, provided herein is a composition comprising antigens from other infectious organisms. Other examples of infectious organisms include, but are not limited to, protozoan parasites, such as Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Trichinella spiralis, Strongyloides ratti, and Toxoplasma gondii.

[0090] Also provided herein are compositions for treating cancer. In some embodiments, the compositions comprise RNA encoding tumor virus antigens. In some embodiments, the tumor virus antigens are from human papillomavirus (HPV) antigens, Kaposi Sarcoma (Kaposi Sarcoma) associated herpes virus (KSHV) antigens, Merkel cell polyomavirus (MCV) antigens, human T-cell lymphotropic virus type 1 (HTLV-1) antigens, or Epstein-Barr virus (EBV) antigens.

[0091] In some embodiments, the compositions provided herein include RNA encoding tumor antigens. In some embodiments, tumor antigens are surface proteins, cytoplasmic proteins or transmembrane proteins. Tumor antigens are proteins expressed by cancer cells. Some tumor antigens (referred to as neoantigens) are proteins produced in cancer cells with point mutations, frameshift mutations and gene rearrangements. The RNA molecules provided herein can encode a tumor antigen or multiple tumor antigens. Non-limiting examples of tumor antigens include mucin (e.g., MUC1), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (Her2 / neu), telomerase (TERT), survivin, melanoma-associated antigen A1 (MAGE-A1), MAGE-A3, Wilms' tumor 1 (WT1), melanoma preferentially expressed antigen (PRAME), New York esophagus 1 (NY-ESO-1), tumor protein p53, tyrosinase, S100 protein, melanoma-associated antigen (MART-1) recognized by T cells 1 or any combination thereof.

[0092] In addition, the RNA molecules provided herein may encode proteins that regulate the immune response to cancer cells or tumors. In some embodiments, the compositions provided herein include RNA encoding immunomodulatory proteins. In some embodiments, the compositions include one or more RNA sequences encoding immunomodulatory proteins. In some embodiments, the immunomodulatory proteins are CD83, 4-1BB ligands, cytokines, or any combination thereof. Exemplary cytokines for treating cancer include, but are not limited to, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-14, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, CCL3, CCL5, and CXCR4.

[0093] The compositions provided herein may also include a delivery vehicle. Non-limiting examples of delivery vehicles that can be used include: solvents, diluents, aqueous solutions, saline solutions, particles, emulsifiers, surfactants, lipid carriers, lipids, engineered vesicles, cells, extracellular vesicles, yeast cells, antibodies, aptamers, vectors, viral vectors, adenoviral vectors, lentiviral vectors, micelles, liposomes, and transfection agents (e.g., multivalent cationic lipid compositions, such as polyethyleneimine (PEI), LIPOFECTAMINE TM , DMRIE-C, DMRIE, DOTAP, DOSPA and DOSPER and dendrimer compositions, particularly G5-G10 dendrimers, including dense star dendrimers, PAMAM dendrimers, grafted dendrimers and dendrimers known as dendrigrafts and ).

[0094] Provided herein is a vector, wherein the vector comprises: one or more nucleic acids encoding: a TLR5 agonist provided herein, a TLR4 agonist provided herein, a TLR2 agonist provided herein, or a combination thereof. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus-like particle. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises adenovirus, adeno-associated virus (AAV), recombinant AAV (rAAV), slow virus, alpha virus, flavivirus, rhabdovirus, herpes simplex virus, Kunjin virus (Kunjin virus), measles virus, Lassa virus, or its virus-like particle. In some embodiments, AAV or rAAV comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAV10rh. In some embodiments, the viral vector is a self-replicating viral vector (e.g., alpha virus) or a portion thereof.

[0095] In some embodiments, one or more nucleic acids comprise DNA. In some embodiments, one or more nucleic acids comprise RNA. In some embodiments, the vector comprises a DNA sequence encoding an RNA sequence having at least 85% identity with any one of SEQ ID NOs: 2 to 37. In some embodiments, the vector comprises a sequence having at least 85% identity with SEQ ID NO: 226. In some embodiments, the vector comprises a nucleic acid sequence having at least 85% identity with any one of SEQ ID NOs: 2 to 37. In some embodiments, the vector comprises a DNA sequence encoding an RNA sequence having at least 85% identity with any one of SEQ ID NOs: 38 to 103. In some embodiments, the vector comprises a nucleic acid sequence having at least 85% identity with any one of SEQ ID NOs: 38 to 103. In some embodiments, the vector comprises a DNA sequence encoding an RNA sequence having at least 85% identity with any one of SEQ ID NOs: 104 to 175. In some embodiments, the vector comprises a nucleic acid sequence having at least 85% identity with any one of SEQ ID NOs: 104 to 175. In some embodiments, the vector comprises a nucleic acid sequence encoding a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen. In some embodiments, the vector comprises a sequence encoding a SARS-CoV-2 spike protein or any antigen provided herein.

[0096] Liposomes and / or nanoparticles can also be used with the administration of the compositions herein. Liposomes are formed by phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also referred to as multilamellar vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Ultrasonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 angstroms to 500 angstroms, containing an aqueous solution in the core.

[0097] Nanoparticle carriers can also be used as delivery vehicles for RNA molecules provided herein. In some embodiments, nanoparticles are gold nanoparticles, platinum nanoparticles, iron oxide nanoparticles, lipid nanoparticles, selenium nanoparticles, glycol chitosan nanoparticles (CNP), cathepsin B sensitive nanoparticles, hyaluronic acid nanoparticles, paramagnetic nanoparticles or polymer nanoparticles. Additional carriers and nanoparticles will be discussed further below.

[0098] (3) Carrier

[0099] Provided herein are compositions comprising carriers. Carriers help deliver nucleic acids provided herein to cells or tissues. In some embodiments, carriers are lipid carriers or lipid nanoparticles. In some embodiments, nucleic acids provided herein (e.g., DNA, RNA or RNA adjuvants provided herein) are compounded with lipid carriers. In some embodiments, nucleic acids provided herein (e.g., DNA, RNA or RNA adjuvants provided herein) are encapsulated in lipid carriers. In some embodiments, nucleic acids encoding antigens provided herein are compounded with lipid carriers. In some embodiments, nucleic acids encoding antigens provided herein are encapsulated in lipid carriers. In some embodiments, carriers provided herein are compounded with lipid carriers. In some embodiments, carriers provided herein are encapsulated in lipid carriers. In some embodiments, protein antigens provided herein are compounded with lipid carriers. In some embodiments, protein antigens provided herein are encapsulated in lipid carriers. In some embodiments, nucleic acids encoding antigens and RNA adjuvants, TLR agonists or nucleic acids encoding TLR agonists are encapsulated in the same lipid carrier. In some embodiments, RNA encoding antigens and RNA adjuvants are encapsulated in different lipid carriers. Exemplary configurations of carrier-RNA complexes are provided in FIG. 2A to FIG. 2D middle.

[0100] In some embodiments, the lipid carriers provided herein include cationic lipids, polyethylene glycol (PEG) modified lipids, sterols, and non-cationic lipids. In some embodiments, the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a PEG lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a liposome complex, a lipid class, a derivative, a variant, or any combination thereof.

[0101] In some embodiments, the lipid carrier comprises a cationic lipid. In some embodiments, the lipid carrier provided herein comprises an ionizable cationic lipid. In some embodiments, the cationic lipid is selected from the group consisting of: 2,2-dioleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dioleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-ene-1-yl) 9-((4-(dimethylamino)butyryl)oxy) heptadecanedioate (L319). In some embodiments, the molar ratio of the cationic lipid carrier is about 20%-60% cationic lipid: about 5%-25% non-cationic lipid: about 25%-55% sterol; and about 0.5%-15% PEG-modified lipid. In some embodiments, the cationic lipid carrier comprises a molar ratio of about 50% cationic lipid, about 1.5% PEG-modified lipid, about 38.5% cholesterol and about 10% non-cationic lipid. In some embodiments, the cationic lipid carrier comprises a molar ratio of about 55% cationic lipid, about 2.5% PEG lipid, about 32.5% cholesterol and about 10% non-cationic lipid. In some embodiments, the cationic lipid carrier is an ionizable cationic lipid, and the non-cationic lipid is a neutral lipid, and sterol is cholesterol. In some embodiments, the cationic lipid nanoparticle has a molar ratio of about 50:38.5:10:1.5 cationic lipid: cholesterol: PEG2000-DMG: DSPC. In some embodiments, the cationic lipid carrier has an average diameter of about 50 nanometers (nm) to about 200nm. In some embodiments, the cationic lipid nanoparticle has an average diameter of about 80nm to 100nm. The compositions provided herein may include about 2mg / mL or less RNA and about 30mg / mL to about 50mg / mL lipid.

[0102] In some embodiments, the lipid carrier provided herein comprises cholesterol. In some embodiments, the lipid carrier provided herein comprises PEGylated lipids. In some embodiments, the lipid carrier provided herein comprises distearoylphosphatidylcholine (DSPC). In some embodiments, the lipid carrier provided herein comprises ionizable cationic lipids, cholesterol, PEGylated lipids and distearoylphosphatidylcholine (DSPC).

[0103] Lipid nanoparticle (LNP) formulations can also include coatings with surfactants or polymers to improve the delivery of nanoparticles. In some embodiments, nanoparticles can be coated with hydrophilic coatings, such as but not limited to PEG coatings and / or coatings with neutral surface charge. Hydrophilic coatings can help deliver nanoparticles with larger payloads. In some embodiments, lipid nanoparticles include hydrophilic polymer particles.

[0104] Nanoparticle formulations provided herein can also include carbohydrate nanoparticles, which include carbohydrate carriers and RNA encoding antigens and / or RNA adjuvants provided herein. As non-limiting examples, carbohydrate carriers can include, but are not limited to, anhydride-modified phytoglycogen or glycogen-based materials, phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydride-modified phytoglycogen β-dextrin.

[0105] Lipid nanoparticles can also comprise polymeric material (i.e. polymer core) and / or polymer-vitamin conjugate and / or triblock copolymer.Polymeric material can include but is not limited to polyamine, polyether, polyamide, polyester, polyurethane, polyurea, polycarbonate, poly (styrene), polyimide, polysulfone, polyurethane, polyacetylene, polyethylene, polyethyleneimine, polyisocyanate, polyacrylate, polymethacrylate, polyacrylonitrile and polyarylate.Polymeric material can be biodegradable and / or biocompatible.Polymeric material can also be irradiated.As non-limiting example, polymeric material can be irradiated by gamma.Non-limiting examples of specific polymers include polycaprolactone (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-P poly(ethylene glycol), poly(ethylene terephthalate), poly(ethylene glycol ... , polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters (such as poly(vinyl acetate)), polyvinyl halides (such as poly(vinyl chloride)) (PVC), polyvinyl pyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses (such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose), polymers of acrylic acid (such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly((methyl)acrylate) acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof), polydioxanone and copolymers thereof, polyhydroxyalkanoate, polypropylene fumarate, polyoxymethylene, poloxamer, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinyl pyrrolidone.

[0106] The lipid nanoparticles may comprise a surface modifier such as, but not limited to, a polynucleotide, an anionic protein (e.g., bovine serum albumin), a surfactant (e.g., a cationic surfactant such as dimethyldioctadecyl ammonium bromide), a sugar or sugar derivative (e.g., cyclodextrin), a nucleic acid, a polymer (e.g., heparin, polyethylene glycol, and poloxamer), a mucolytic agent (e.g., N-acetylcysteine, artemisia, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbociste ine), eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4 alpha dornase, neltenexine, erdosteine) and various DNA enzymes, including rhDNA enzyme. The surface modifier can be embedded or entrained into the surface of the particle, or arranged (e.g., by coating, adsorption, covalent bonding or other methods) on the surface of the lipid nanoparticle.

[0107] In some embodiments, the carrier is formulated as a liposome complex, such as, but not limited to, ATUPLEX from Silence Therapeutics (London, UK). TM system, DACC system, DBTC system and other siRNA-liposome complex technologies; from STEMFECT (Cambridge, Massachusetts) TM ; and polyethyleneimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids.

[0108] In some embodiments, the compositions provided herein are formulated into solid lipid nanoparticles. Solid lipid nanoparticles (SLN) can be spherical with an average diameter between 10nm and 1000nm. SLN has a solid lipid core matrix that can dissolve lipophilic molecules, and can be stabilized with surfactants and / or emulsifiers. The type of lipid carrier used in the compositions provided herein will depend on the target site, mode of administration, and RNA delivery to the target site to induce an immune response in a subject.

[0109] (4) Yeast cell composition

[0110] Provided herein is a composition comprising: (a) a yeast cell, wherein the yeast cell comprises a permeable cell wall; and (b) one or more nucleic acids (e.g., an RNA adjuvant), wherein the one or more nucleic acids encode at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. In some embodiments, the one or more nucleic acids (e.g., an RNA adjuvant) are encapsulated in a yeast cell. In some embodiments, the one or more nucleic acids (e.g., an RNA adjuvant) are compounded with a yeast cell.

[0111] In order to prepare a composition comprising yeast cells as a delivery vehicle or carrier, the yeast cells can be contacted with a permeabilizing agent before compounding or encapsulating a nucleic acid provided herein (e.g., an RNA adjuvant encoding a TLR agonist). In some embodiments, the permeabilizing agent is a β-glucanase. In some embodiments, the β-glucanase is a b-1-3-glucanase. In some embodiments, the yeast cell is a Pichia pastoris cell, a Saccharomyces cerevisiae cell, or a Kluyveromyces lactis cell.

[0112] In some embodiments, the compositions provided herein further comprise a nucleic acid (e.g., RNA sequence) encoding an antigen provided herein (e.g., a viral antigen, e.g., a SARS-CoV-2 spike protein or a fragment thereof). In some embodiments, the nucleic acid provided herein comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acid provided herein comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acid provided herein comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acid provided herein comprises any one of SEQ ID NOs: 2 to 175. In some embodiments, the nucleic acid provided herein encodes an amino acid sequence selected from any one of SEQ ID NOs: 170 to 210. In some embodiments, the compositions provided herein further comprise enteric coated microspheres. In some embodiments, the yeast cells are encapsulated in enteric coated microspheres.

[0113] In some embodiments, the compositions provided herein are formulated for oral administration to a subject. In some embodiments, the compositions comprising yeast cells provided herein are mixed with food compositions. In some embodiments, when food compositions are prepared or formulated into functional foods (e.g., nutritional supplements). In other embodiments, such compositions can be prepared or formulated into, for example, medicines, dietary supplements and / or medical foods.

[0114] (5) Pharmaceutical vaccine composition, dosage and administration

[0115] Provided herein are pharmaceutical compositions and vaccine compositions comprising one or more nucleic acids provided herein or vectors provided herein; and pharmaceutically acceptable diluents, carriers, or excipients. Also provided herein are pharmaceutical compositions or vaccine compositions comprising two or more of the following: (a) nucleic acids encoding TRL agonists (e.g., RNA adjuvants); (b) nucleic acid sequences encoding antigens (e.g., RNA sequences); and / or (c) carriers; and (d) pharmaceutically acceptable diluents, carriers, or excipients. Also provided herein are pharmaceutical compositions comprising carriers comprising or encoding any one of the nucleic acids provided herein (e.g., RNA adjuvants) or proteins; and pharmaceutically acceptable diluents, carriers, or excipients.

[0116] In some embodiments, the compositions provided herein (e.g., RNA adjuvants) are combined with pharmaceutically acceptable salts, excipients and / or carriers to form pharmaceutical compositions. Pharmaceutical salts, excipients and carriers can be selected according to the route of administration, the location of the target tissue and the time course of drug delivery. Pharmaceutically acceptable carriers or excipients may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents compatible with drug administration, etc.

[0117] In some embodiments, the pharmaceutical composition is in the form of solid, semisolid, liquid or gas (aerosol). Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents. In acceptable vehicles and solvents, water, Ringer's solution, USP and isotonic sodium chloride solution can be used. In addition, sterile, non-volatile oils are generally used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injectables. Injectable formulations can be sterilized, for example, by filtering through a bacterial retention filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0118] For ease of administration and uniformity of dosage, RNA adjuvants, nucleic acids, compositions, vaccine compositions, and pharmaceutical compositions for administration to subjects in need thereof can be formulated in unit dosage form. Unit dosage forms are physically discrete units of the compositions provided herein, suitable for subjects to be treated. However, it should be understood that the total dosage of the compositions provided herein will be determined by the attending physician within the scope of reasonable medical judgment. For any composition provided herein, the therapeutically effective dose can be initially estimated in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. Animal models are also used to obtain the ideal concentration range and route of administration. Such information can then be used to determine the effective dose and route for human administration. 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 (This dose is lethal to 50% of the population.) The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 In some embodiments, pharmaceutical compositions that exhibit large therapeutic indices may be useful. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.

[0119] Provided herein are nucleic acids, RNA adjuvants, compositions, vaccine compositions, and pharmaceutical compositions for administration to subjects in need thereof (e.g., as vaccines or as treatments for infectious diseases or cancer). In some embodiments, the administration of the compositions provided herein is topical administration. In some embodiments, the administration of the compositions provided herein is systemic administration. In some embodiments, the compositions provided herein are formulated for administration by subcutaneous, intradermal, intramuscular, inhaled, intravenous, intraperitoneal, or oral routes. In some cases, the treatment regimen can be administered according to the subject's weight. For example, a body mass index can be used. BMI is calculated by: BMI=weight (kg) / [height (m)] 2 .

[0120] By way of example only, exemplary dosages of the nucleic acid and RNA vaccines provided herein are provided below. In some embodiments, nucleic acids encoding RNA adjuvants or TLR agonists provided herein and / or nucleic acids encoding antigens provided herein are administered in an amount of at least about 1 nanogram (ng) or more, about 5 ng or more, about 10 ng or more, about 20 ng or more, about 30 ng or more, about 40 ng or more, about 50 ng or more, about 60 ng or more, about 70 ng or more, about 80 ng or more, about 90 ng or more, 100 ng or more, 110 ng or more, 120 ng or more, 130 ng or more, 140 ng or more, 150 ng or more, 160 ng or more, 170 ng or more, 180 ng or more, 190 ng or more, 200 ng or more, 210 ng or more, 220 ng or more, 230 ng or more, 240 ng or more, 250 ng or more, 260 ng or more, 270 ng or more, 280 ng or more, 290 ng or more, 300 ng or more, 350 ng or more, 400 ng or more, up to 500 ng. In some embodiments, the nucleic acid provided herein is administered in an amount of at least about 1 nanogram (ng) or more, 10 ng or more, 50 ng or more, 100 ng or more, 150 ng or more, 200 ng or more, 250 ng or more, 500 ng or more, or 1 (μg) microgram. In some embodiments, the nucleic acid provided herein is administered in an amount of at least about 1 microgram (μg) or more, about 5 μg or more, about 10 μg or more, about 20 μg or more, about 30 μg or more, about 40 μg or more, about 50 μg or more, about 60 μg or more, about 70 μg or more, about 80 μg or more, about 90 μg or more, up to 100 μg.

[0121] In some embodiments, the compositions provided herein are administered intravenously or intramuscularly. In some embodiments, the compositions provided herein are administered at least about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 2000 mg / kg, about 3000 mg / kg, about 4000 mg / kg, about 5000 mg / kg, about 6000 mg / kg, about 7000 mg / kg, about 8000 mg / kg, about 9000 mg / kg, about 10000 mg / kg, about 20000 mg / kg, about 30000 mg / kg, about 40000 mg / kg, about 50000 mg / kg, about 50000 mg / kg, about 60000 mg / kg, about 70000 mg / kg, about 80000 mg / kg, about 90000 mg / kg, about 100000 mg / kg, about 200000 mg / kg, about 300000 mg / kg, about 40000 mg / kg, about 500000 mg / kg, about mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 2000 mg / kg, about 2200 mg / kg, about 2400 mg / kg, up to a concentration of about 2500 mg / kg.

[0122] In some embodiments, the compositions provided herein are administered orally. In some embodiments, the compositions provided herein are administered orally at a dosage of at least about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about g / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 2000 mg / kg, about 2200 mg / kg, about 2400 mg / kg, up to a concentration of about 2500 mg / kg orally administered.

[0123] In some embodiments, the dosage of the compositions provided herein can be administered once a day, or divided into sub-doses and administered in multiple doses, such as daily, twice a day, weekly, biweekly, monthly, bimonthly, annually, etc. In some embodiments, administration is repeated at least about every 1 year (8760 hours, 365 days), 2 years, 3 years, 4 years, 5 years or longer. In some embodiments, an additional dose of nucleic acid, pharmaceutical composition or vaccine composition is administered to the subject. In some embodiments, administration includes administering a pharmaceutical composition of a first dose and administering a pharmaceutical composition of a second dose at least about 2 weeks after the first dose. In some embodiments, the composition is a booster dose vaccine composition, which is a composition of a second dose, a third dose or a fourth dose administered to a subject.

[0124] RNA adjuvant and RNA vaccine composition (for example, RNA encoding antigen) can be applied simultaneously or sequentially in the same composition. The RNA encoding TLR agonist provided herein can be on the same nucleic acid chain as the RNA sequence encoding antigen, or on different nucleic acid chains. When applied sequentially, the time between applications is selected to facilitate the efficacy and / or safety of combined treatments, etc. In certain embodiments, the RNA adjuvant composition provided herein can be applied first, and then the RNA vaccine is applied, or alternatively, the RNA vaccine is applied first, and then the RNA adjuvant composition of the present disclosure (for example, a composition comprising the RNA sequence listed in Table 1) is applied. As an example and not limitation, the time between applications is about 1 hour (hr), about 2 hours (hrs), about 4hrs, about 6hrs, about 12hrs, about 16hrs or about 20hrs. In certain embodiments, the time between applications is about 24 hours (1 day), about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days or more. In some embodiments, the time between applications is about 1 week (7 days), 2 weeks, 3 weeks or 4 weeks or longer. In some embodiments, the time between administrations is about 1 month (30 days), 2 months (60 days) or longer. When administered simultaneously, the RNA adjuvant composition can be administered separately with the second RNA vaccine by the same or different routes, or administered in a single pharmaceutical composition by the same route. In certain embodiments, the dosage and frequency of administration of the RNA vaccine can use the standard dose and standard frequency of administration for the specific RNA vaccine. In some embodiments, when administered with the RNA adjuvant composition provided herein, the dosage of the RNA vaccine is reduced. This is due to the enhancement of the innate immune response by the RNA adjuvant-RNA vaccine combination.

[0125] (6) Therapeutic applications

[0126] The compositions and adjuvants provided herein are formulated for treating a disease or condition. Also provided herein is a method for treating a disease or condition in a subject. In some embodiments, the disease or condition is cancer or infection. The adjuvants provided herein can be administered to a subject alone or in combination with an additional agent (e.g., a chemotherapeutic agent, a vaccine, an antiviral drug, or an immunotherapy) in the same composition or a separate composition.

[0127] Provided herein is a method for treating a disease or condition in a subject, comprising: administering to the subject a medicament in combination with an adjuvant provided herein. In some embodiments, the medicament is administered prior to administering the adjuvant. In some embodiments, the medicament is administered simultaneously with the adjuvant. In some embodiments, the medicament is administered after administering the adjuvant. In some embodiments, provided herein is a composition comprising a medicament and an adjuvant in a unit dosage form.

[0128] In some embodiments, after administering an effective amount of an agent or composition provided herein to a subject in the presence of an antigen, an initial innate immune response is induced in the subject. In some embodiments, the innate immune response comprises an increase in the level or activity of nuclear factor κB (NF-κB) in epithelial cells of the subject relative to the level or activity of NF-κB in the absence of the composition.

[0129] In some embodiments, the subject suffers from infection, is diagnosed with infection, or is at risk of developing infection. In some embodiments, infection is a viral infection. In some embodiments, viral infection is an upper respiratory tract viral infection. In some embodiments, upper respiratory tract viral infection is COVID-19, SARS infection, MERS infection, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia or rhinovirus infection. In some embodiments, infection is a bacterial infection, a fungal infection, a parasitic infection or a yeast infection. In some embodiments, the subject is infected with an infectious disease by contacting with another infected subject. In some embodiments, the subject is infected with an infectious disease from different species carrying microorganisms. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a person.

[0130] Provided herein are methods of stimulating an immune response in a subject, the methods comprising: administering to the subject an amount of a composition provided herein sufficient to stimulate the immune response.

[0131] Provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, the method comprising: (a) administering to a subject an effective amount of a composition provided herein or a vaccine composition provided herein; and (b) administering to the subject an RNA vaccine composition, wherein the RNA vaccine composition comprises RNA encoding the antigen, thereby enhancing the immune response to the antigen encoded by the RNA.

[0132] In some embodiments, the method further comprises administering an RNA vaccine composition to the subject. In some embodiments, the method further comprises administering a protein antigen vaccine composition to the subject. In some embodiments, the antigen is a spike protein, a glycoprotein, a viral envelope protein, or a cancer cell protein. In some embodiments, the spike protein is from a SARS-CoV-2 virus. In some embodiments, the composition induces an immune response to a viral antigen, wherein the viral antigen is selected from adenovirus, astrovirus, bocavirus, Bannavirus, BK virus, Colti virus, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barr virus, hantavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus (HIV), influenza virus, JC virus, Lassa virus, Marburg virus, measles virus, Middle East respiratory syndrome (MERS) coronavirus, Norwalk virus, Norovirus, parvovirus, papillomavirus, poliovirus, rabies virus, rhinovirus, rotavirus, rubella virus, severe acute respiratory syndrome (SARS) virus, smallpox virus, varicella-zoster virus, yellow fever virus, or any combination thereof.

[0133] Also provided herein is a method for treating a subject's cancer, wherein the method includes administering a composition provided herein, so as to treat cancer. In some embodiments, the subject suffers from cancer, is diagnosed with cancer, or is at risk of developing cancer. In some embodiments, administration is intratumoral administration, intrasplenic administration, intradermal administration, intranodal administration, intramuscular administration, oral administration, intranasal administration, or intravenous administration. In some embodiments, the subject suffers from a solid tumor or blood cancer. In some embodiments, the subject suffers from the following cancers, is suspected of having the following cancers, or is diagnosed with the following cancers: prostate cancer, ovarian cancer, breast cancer, lung cancer, leukemia, brain cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, soft tissue sarcoma, skin cancer, gastric cancer, thyroid cancer, or uterine cancer. In some embodiments, the subject suffers from a solid tumor. In some embodiments, the subject suffers from metastatic cancer. In some embodiments, the composition for treating cancer comprises RNA or DNA encoding a tumor antigen provided herein. In some embodiments, a composition for treating cancer comprises RNA encoding an immunomodulatory protein provided herein.

[0134] Exemplary embodiments

[0135] Provided herein are compositions for inducing an immune response after administration to a subject, wherein the composition comprises: (a) an RNA adjuvant encoding a protein, wherein the protein comprises at least one functional fragment of a toll-like receptor 5 (TLR5) agonist, wherein the functional fragment activates the TLR5 pathway in the cell when in contact with the cell; and (b) one or more delivery vehicles, wherein after administering an effective amount of the composition to the subject in the presence of an antigen, an initial innate immune response is induced in the subject. Also provided herein are compositions, wherein one or more delivery vehicles are formulated for intranasal administration, oral administration, intradermal administration, intranodal administration, intrasplenic administration, intratumoral administration, intravenous administration, or intramuscular administration. Also provided herein are compositions, wherein the functional fragment of the TLR5 agonist activates the TLR5 pathway in the cell when in contact with the cell. Also provided herein are compositions, wherein the cell is an epithelial cell. Also provided herein are compositions, wherein the functional fragment of the TLR5 agonist comprises a functional derivative of a bacterial movement protein. Also provided herein are compositions, wherein the bacterial movement protein is a bacterial flagellin, a derivative thereof, or a functional fragment thereof. Also provided herein is a composition, wherein the protein is a truncated form of bacterial flagellin, a secreted form of bacterial flagellin, or a combination thereof. Also provided herein is a composition, wherein the functional fragment of a toll-like receptor 5 (TLR5) agonist comprises the D0 region and the D1 region of bacterial flagellin. Also provided herein is a composition, wherein the functional fragment of a toll-like receptor 5 (TLR5) agonist comprises the D0 region, the D1 region, and the D2 region of bacterial flagellin. Also provided herein is a composition, wherein the functional fragment of a toll-like receptor 5 (TLR5) agonist comprises the D0 region, the D1 region, the D2 region, and the D3 region of bacterial flagellin. Also provided herein is a composition, wherein the synthetic protein further comprises a signal sequence. Also provided herein is a composition, wherein the signal sequence comprises the following amino acid sequence: MRSLSVLALLLLLLLAPASA (SEQ ID NO: 1). Also provided herein is a composition, wherein the RNA adjuvant comprises a sequence having at least 85% identity to any one of SEQ ID NO: 2 to 37. Also provided herein are compositions for inducing an immune response, wherein the RNA adjuvant comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 2 to 37. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 2 to 37. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 96% identity to any one of SEQ ID NOs: 2 to 37. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 97% identity to any one of SEQ ID NOs: 2 to 37. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 98% identity to any one of SEQ ID NOs: 2 to 37.Compositions are also provided herein, wherein the RNA adjuvant comprises a sequence having at least 99% identity to any one of SEQ ID NO: 2 to 37. Compositions are also provided herein, wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NO: 2 to 37. Compositions are also provided herein, wherein one or more delivery vehicles comprise a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a carrier, or any combination thereof. Compositions are also provided herein, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen. Compositions are also provided herein, wherein the antigen is a tumor antigen. Compositions are also provided herein, wherein the composition further comprises an RNA encoding an antigen, wherein the RNA encoding the antigen and the RNA adjuvant are in the same delivery vehicle. Compositions are also provided herein, wherein the composition further comprises an RNA encoding an antigen, wherein the RNA encoding the antigen and the RNA adjuvant are in different delivery vehicles. Compositions are also provided herein, wherein the RNA adjuvant further comprises an RNA sequence encoding an antigen. Compositions are also provided herein, wherein the RNA sequence encoding the antigen is operably linked to the RNA adjuvant. Also provided herein are compositions, wherein the innate immune response comprises an increase in the level or activity of nuclear factor κB (NF-κB) in epithelial cells of the subject relative to the level or activity of NF-κB in the absence of the composition.

[0136] Provided herein are compositions for inducing an immune response after administration to a subject, wherein the composition comprises: (a) a nucleic acid sequence encoding a protein, wherein the protein comprises at least one functional fragment of a toll-like receptor 5 (TLR5) agonist, wherein the functional fragment activates the TLR5 pathway in the cell when in contact with the cell; and (b) one or more delivery vehicles, wherein after administering an effective amount of the composition to the subject in the presence of an antigen, an initial innate immune response is induced in the subject. Also provided herein are compositions, wherein the nucleic acid sequence comprises an RNA sequence or a DNA sequence. Also provided herein are compositions, wherein the protein is a synthetic protein.

[0137] Provided herein are compositions, wherein the composition comprises: (a) a lipid carrier; and (b) an RNA sequence encoding at least one functional fragment of a bacterial movement protein, wherein the RNA adjuvant is a TLR5 agonist. Also provided herein are compositions, wherein the RNA adjuvant is encapsulated in a lipid carrier. Also provided herein are compositions, wherein the RNA adjuvant is complexed with a lipid carrier. Also provided herein are compositions, wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipid complex, a lipid class, a derivative, a variant, or any combination thereof. Also provided herein are compositions, wherein at least one functional fragment of a bacterial movement protein is a bacterial flagellin, a derivative thereof, or a functional fragment thereof. Also provided herein are compositions, wherein the bacterial movement protein is a truncated form of a bacterial flagellin, a secreted form of a bacterial flagellin, or a combination thereof. Also provided herein are compositions, wherein the functional fragments of the bacterial movement protein comprise the D0 region and the D1 region of the bacterial flagellin. Also provided herein is a composition, wherein the functional fragment of the bacterial movement protein comprises the D0 region, D1 region and D2 region of the bacterial flagellin. Also provided herein is a composition, wherein the functional fragment of the bacterial movement protein comprises the D0 region, D1 region, D2 region and D3 region of the bacterial flagellin. Also provided herein is a composition, wherein the RNA adjuvant comprises mRNA. Also provided herein is a composition, wherein the composition also comprises one or more RNA molecules each encoding an antigen. Also provided herein is a composition, wherein the composition also comprises one or more antigens. Also provided herein is a composition, wherein the RNA encoding the antigen is mRNA. Also provided herein is a composition, wherein the mRNA is chemically modified. Also provided herein is a composition, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase or a 5' end cap. Also provided herein is a composition, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen or a tumor antigen. Compositions are also provided herein, wherein the viral antigens are from adenovirus, astrovirus, bocavirus, Banna virus, BK virus, Colti virus, coronavirus, coxsackie virus, cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barr virus, Hanta virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus (HIV), influenza virus, JC virus, Lassa virus, Marburg virus, measles virus, Middle East respiratory syndrome (MERS) coronavirus, Norwalk virus, Norovirus, parvovirus, papillomavirus, poliovirus, rabies virus, rhinovirus, rotavirus, rubella virus, severe acute respiratory syndrome (SARS) virus, smallpox virus, varicella zoster virus, yellow fever virus, or any combination thereof. Compositions are also provided herein, wherein the SARS virus is SARS-CoV-2 virus.Compositions are also provided herein, wherein the antigen comprises a spike protein, a glycoprotein, or a hemagglutinin protein. Compositions are also provided herein, wherein the RNA encoding the antigen and the RNA adjuvant are encapsulated in the same lipid carrier. Compositions are also provided herein, wherein the RNA encoding the antigen and the RNA adjuvant are encapsulated in different lipid carriers. Compositions are also provided herein, wherein the RNA adjuvant comprises a sequence having at least 85% identity with any one of SEQ ID NO: 2 to 37. Compositions are also provided herein, wherein the RNA adjuvant comprises a sequence having at least 90% identity with any one of SEQ ID NO: 2 to 37. Compositions are also provided herein, wherein the RNA adjuvant comprises a sequence having at least 95% identity with any one of SEQ ID NO: 2 to 37. Compositions are also provided herein, wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NO: 2 to 37. Compositions are also provided herein, wherein the composition is in the form of a suspension, an aqueous solution, or an emulsion.

[0138] Provided herein are compositions, wherein the compositions comprise: (a) a carrier; and (b) a nucleic acid encoding at least one functional fragment of a bacterial movement protein, wherein the functional fragment of the bacterial movement protein is a TLR5 agonist. Also provided herein are compositions, wherein the carrier is a lipid carrier. Also provided herein are compositions, wherein the nucleic acid comprises RNA, DNA, or a combination thereof.

[0139] Provided herein are compositions for inducing an immune response after administration to a subject, wherein the composition comprises: (a) an RNA adjuvant encoding a protein, wherein the protein comprises at least one functional fragment of a toll-like receptor 4 (TLR4) agonist; and (b) one or more delivery vehicles, wherein the one or more delivery vehicles are formulated for intranasal administration, oral administration, intradermal administration, intranodal administration, intrasplenic administration, intratumoral administration, intravenous administration or intramuscular administration, wherein after administering an effective amount of the composition to the subject in the presence of an antigen, an initial innate immune response is induced in the subject. Also provided herein are compositions for inducing an immune response, wherein the TLR4 agonist comprises at least one functional fragment of a high mobility group protein B1 (HMGB1) protein, nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, a transglycosylase, a 50S ribosomal protein, a heparin-binding hemagglutinin protein, a YadA family autotransporting adhesin, a dnaJ protein, a pneumolysin protein, an Omp19 protein, a 6,7-dimethyl-8-ribosylcytosine synthase, or any combination thereof. TLR4 agonists are from bacteria. Also provided herein are compositions for inducing an immune response, wherein the TLR4 agonist is from humans. Also provided herein are compositions for inducing an immune response, wherein the RNA comprises a sequence having at least 85% identity to any one of SEQ ID NO:38 to 103. Also provided herein are compositions for inducing an immune response, wherein the RNA comprises a sequence having at least 90% identity to any one of SEQ ID NO:38 to 103. Also provided herein are compositions for inducing an immune response, wherein the RNA comprises a sequence having at least 95% identity to any one of SEQ ID NO:38 to 103. Also provided herein are compositions for inducing an immune response, wherein the RNA comprises a sequence selected from any one of SEQ ID NO:38 to 103. Also provided herein are compositions for inducing an immune response, wherein the delivery vehicle is a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, or any combination thereof. Also provided herein are compositions for inducing an immune response, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, or a tumor antigen. Also provided herein are compositions for inducing an immune response, wherein the innate immune response comprises an increase in the level or activity of NF-κB when the composition is administered relative to the level or activity of NF-κB in the absence of the composition.

[0140] Provided herein are compositions, wherein the compositions comprise: (a) a lipid carrier; and (b) an RNA sequence encoding at least one functional fragment of a TLR4 agonist. Also provided herein are compositions, wherein the TLR4 agonist comprises at least one functional fragment of a high mobility group protein B1 (HMGB1) protein, a nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, a transglycosylase, a 50S ribosomal protein, a heparin-binding hemagglutinin protein, a YadA family autotransporting adhesin, a dnaJ protein, a pneumolysin protein, an Omp19 protein, a 6,7-dimethyl-8-ribosylcytosine synthase, or any combination thereof. Also provided herein are compositions, wherein the TLR4 agonist is from bacteria. Also provided herein are compositions, wherein the TLR4 agonist is from humans. Also provided herein are compositions, wherein the RNA comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 38 to 103. Also provided herein are compositions, wherein the RNA comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 38 to 103. Compositions are also provided herein, wherein the RNA comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 38 to 103. Compositions are also provided herein, wherein the RNA comprises a sequence selected from any one of SEQ ID NOs: 38 to 103. Compositions are also provided herein, wherein the RNA adjuvant is encapsulated in a lipid carrier. Compositions are also provided herein, wherein the RNA adjuvant is complexed with a lipid carrier. Compositions are also provided herein, wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipid complex, a lipid class, a derivative, a variant, or any combination thereof. Compositions are also provided herein, wherein the RNA adjuvant comprises mRNA. Compositions are also provided herein, wherein the composition further comprises one or more RNA molecules each encoding an antigen. Compositions are also provided herein, wherein the composition further comprises one or more antigens. Compositions are also provided herein, wherein the RNA encoding the antigen is mRNA. Compositions are also provided herein, wherein the mRNA is chemically modified. Also provided herein are compositions wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap. Also provided herein are compositions wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.Compositions are also provided herein, wherein the viral antigens are from adenovirus, astrovirus, bocavirus, Banna virus, BK virus, Colti virus, coronavirus, coxsackie virus, cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barr virus, Hanta virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus (HIV), influenza virus, JC virus, Lassa virus, Marburg virus, measles virus, Middle East respiratory syndrome (MERS) coronavirus, Norwalk virus, Norovirus, parvovirus, papillomavirus, poliovirus, rabies virus, rhinovirus, rotavirus, rubella virus, severe acute respiratory syndrome (SARS) virus, smallpox virus, varicella zoster virus, yellow fever virus, or any combination thereof. Compositions are also provided herein, wherein the SARS virus is a SARS-CoV-2 virus. Compositions are also provided herein, wherein the antigen comprises a spike protein, a glycoprotein, a hemagglutinin protein, or a cancer cell protein. Compositions are also provided herein, wherein the RNA encoding the antigen and the RNA adjuvant are encapsulated in the same lipid carrier. Compositions are also provided herein, wherein the RNA encoding the antigen and the RNA adjuvant are encapsulated in different lipid carriers. Compositions are also provided herein, wherein the composition is in the form of a suspension, aqueous solution or emulsion.

[0141] Compositions are provided herein, wherein the composition comprises: (a) a carrier; and (b) a nucleic acid encoding at least one functional fragment of a TLR4 agonist. Compositions are also provided herein, wherein the carrier is a lipid carrier. Compositions are also provided herein, wherein the nucleic acid comprises RNA, DNA, or a combination thereof.

[0142] Provided herein are compositions for inducing an immune response after administration to a subject, wherein the composition comprises: (a) a RNA adjuvant encoding a protein, wherein the protein comprises at least one functional fragment of a toll-like receptor 2 (TLR2) agonist; and (b) one or more delivery vehicles, wherein after administering an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject. Also provided herein are compositions, wherein one or more delivery vehicles are formulated for intranasal administration, oral administration, intradermal administration, intranodal administration, intrasplenic administration, intratumoral administration, intravenous administration, or intramuscular administration. Also provided herein are compositions, wherein the TLR2 agonist comprises at least one functional fragment of an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system. Also provided herein are compositions, wherein the TLR2 agonist is from bacteria. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the RNA adjuvant comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the RNA comprises a sequence selected from any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the one or more delivery vehicles comprise a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, or any combination thereof. Also provided herein are compositions, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen. Also provided herein are compositions, wherein the innate immune response comprises an increase in the level or activity of NF-kB in a subject when the composition is administered relative to the level or activity of nuclear factor kappa B in the absence of administration of the composition.

[0143] Provided herein are compositions, wherein the composition comprises: (a) a lipid carrier; and (b) an RNA sequence encoding at least one functional fragment of a TLR2 agonist. Also provided herein are compositions, wherein the TLR2 agonist comprises at least one functional fragment of an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system. Also provided herein are compositions, wherein the TLR2 agonist is from bacteria. Also provided herein are compositions, wherein the RNA sequence comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the RNA sequence comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the RNA sequence comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are compositions, wherein the RNA sequence comprises a sequence selected from any one of SEQ ID NOs: 104 to 175. Compositions are also provided herein, wherein the RNA sequence is encapsulated in a lipid carrier. Compositions are also provided herein, wherein the RNA sequence is compounded with a lipid carrier. Compositions are also provided herein, wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipid complex, a lipid class, a derivative, a variant, or any combination thereof. Compositions are also provided herein, wherein the RNA sequence comprises mRNA. Compositions are also provided herein, wherein the composition also comprises one or more nucleic acids encoding antigens respectively. Compositions are also provided herein, wherein the composition also comprises one or more antigens. Compositions are also provided herein, wherein the RNA encoding the antigen is mRNA. Compositions are also provided herein, wherein the mRNA is chemically modified. Compositions are also provided herein, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap. Compositions are also provided herein, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, or a tumor antigen.Compositions are also provided herein, wherein the viral antigens are from adenovirus, astrovirus, bocavirus, Banna virus, BK virus, Colti virus, coronavirus, coxsackie virus, cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barr virus, Hanta virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human immunodeficiency virus (HIV), influenza virus, JC virus, Lassa virus, Marburg virus, measles virus, Middle East respiratory syndrome (MERS) coronavirus, Norwalk virus, Norovirus, parvovirus, papillomavirus, poliovirus, rabies virus, rhinovirus, rotavirus, rubella virus, severe acute respiratory syndrome (SARS) virus, smallpox virus, varicella zoster virus, yellow fever virus, or any combination thereof. Compositions are also provided herein, wherein the SARS virus is a SARS-CoV-2 virus. Compositions are also provided herein, wherein the antigen comprises a spike protein, a glycoprotein, a hemagglutinin protein, or a cancer cell protein. Compositions are also provided herein, wherein the RNA sequence encoding the antigen and the RNA sequence are encapsulated in the same lipid carrier. Compositions are also provided herein, wherein the RNA sequence encoding the antigen and the RNA sequence encoding the TLR2 agonist are encapsulated in different lipid carriers. Compositions are also provided herein, wherein the composition is in the form of a suspension, aqueous solution or emulsion.

[0144] Compositions are provided herein, wherein the composition comprises: (a) a carrier; and (b) a nucleic acid encoding at least one functional fragment of a TLR2 agonist. Compositions are also provided herein, wherein the carrier is a lipid carrier. Compositions are also provided herein, wherein the nucleic acid comprises RNA, DNA, or a combination thereof.

[0145] Provided herein are compositions for inducing an immune response after administration to a subject, the compositions comprising: (a) one or more carriers; (b) one or more nucleic acids, wherein the one or more nucleic acids encode: (i) at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or any combination thereof; (ii) a viral antigen; or (iii) a combination of (i) and (ii); and (c) a delivery vehicle, wherein after administration of an effective amount of the composition, an innate immune response is induced in the subject in the presence of a viral antigen, thereby inducing an immune response against the viral antigen. Also provided herein are compositions, wherein the one or more nucleic acids comprise RNA, DNA, or a combination thereof. Also provided herein are compositions, wherein the compositions comprise RNA sequences encoding functional fragments of TLR4 agonists, TLR2 agonists, TLR5 agonists, or any combination thereof. Also provided herein are compositions, wherein the compositions comprise RNA encoding viral antigens. Also provided herein are compositions, wherein the one or more carriers comprise a lipid carrier. Also provided herein are compositions, wherein the delivery vehicle is formulated for intranasal administration, oral administration, intradermal administration, intranodal administration, intrasplenic administration, intratumoral administration, intravenous administration, or intramuscular administration. Also provided herein are compositions, wherein a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a functional fragment of any combination thereof activates a TLR4, TLR2, or TLR5 pathway in a cell when in contact with a cell. Also provided herein are compositions, wherein the cell is an epithelial cell. Also provided herein are compositions, wherein the viral antigen is a spike protein, a glycoprotein, or a viral envelope protein. Also provided herein are compositions, wherein the spike protein is from a SARS-CoV-2 virus. Also provided herein are compositions, wherein the RNA encoding the viral antigen is an mRNA. Also provided herein are compositions, wherein the RNA encoding the viral antigen is chemically modified. Also provided herein are compositions, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap. Also provided herein are compositions, wherein one or more carriers are compounded with one or more RNA adjuvants. Also provided herein are compositions, wherein one or more carriers are compounded with one or more nucleic acids encoding viral antigens. Also provided herein are compositions, wherein one or more nucleic acids are encapsulated by one or more lipid carriers. Also provided herein are compositions, wherein one or more nucleic acids encoding viral antigens are encapsulated in one or more carriers. Compositions are also provided herein, wherein one or more nucleic acids encoding a TLR5 agonist, a TLR4 agonist, or a TLR2 agonist are operably linked to one or more nucleic acids encoding a viral antigen. Compositions are also provided herein, wherein one or more nucleic acids comprise a sequence selected from SEQ ID NO: 2 to 175, or a functional fragment thereof. Compositions are also provided herein, wherein after administering the composition to a subject, the innate immune response induced in the subject is greater than the innate immune response in a subject to which an effective amount of the composition is not administered.Also provided herein are compositions, wherein upon administration of the composition to the subject, the innate immune response induced in the subject is greater than the innate immune response in the subject administered only a composition comprising a nucleic acid encoding a separate viral antigen. Also provided herein are compositions, wherein the innate immune response comprises an increase in the level or activity of NF-κB when an effective amount of the composition is administered relative to the level or activity of nuclear factor κB in the absence of the composition.

[0146] Provided herein are compositions, wherein the composition comprises: (a) an RNA adjuvant comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2 to 175; and (b) a delivery vehicle. Also provided herein are compositions, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0147] Provided herein are compositions, wherein the composition comprises: (a) a DNA sequence encoding an RNA adjuvant, the RNA adjuvant comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2 to 175; and (b) a delivery vehicle. Also provided herein are compositions, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0148] Provided herein are compositions, wherein the composition comprises: (a) a protein comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to any one of SEQ ID NOs: 176 to 217; and (b) a delivery vehicle. Also provided herein are compositions, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0149] Provided herein are compositions, wherein the composition comprises: (a) a DNA sequence encoding a synthetic protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 176 to 217; and (b) a delivery vehicle. Also provided herein are compositions, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

[0150] Provided herein are compositions, wherein the composition comprises: (a) an RNA sequence encoding a synthetic protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 176 to 217; and (b) a delivery vehicle. Also provided herein are compositions, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof. Also provided herein are compositions, wherein the composition comprises one or more nucleic acids, and wherein the one or more nucleic acids comprise RNA, DNA, or a combination thereof. Also provided herein are nucleic acids encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 206. Also provided herein are nucleic acids encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 210. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 212. Also provided herein are nucleic acids encoding an amino acid sequence having at least 85% identity to SEQ ID NO: 213. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 215. Also provided herein are nucleic acids encoding an amino acid sequence that is at least 75% identical to SEQ ID NO:217.

[0151] Compositions are provided herein, wherein the composition comprises: a synthetic protein comprising a protein construct, wherein the protein construct comprises an amino acid sequence selected from SEQ ID NOs: 176 to 217. Compositions comprising two or more, three or more, four or more, or five or more protein constructs are also provided herein. Also provided herein are nucleic acids encoding a protein construct having at least 80% identity to SEQ ID NO: 206. Also provided herein are nucleic acids encoding a protein construct having at least 80% identity to SEQ ID NO: 210. Also provided herein are nucleic acids encoding a protein construct having at least 75% identity to SEQ ID NO: 212. Also provided herein are nucleic acids encoding a protein construct having at least 85% identity to SEQ ID NO: 213. Also provided herein are nucleic acids encoding a protein construct having at least 75% identity to SEQ ID NO: 215. Also provided herein are nucleic acids encoding a protein construct having at least 75% identity to SEQ ID NO: 217.

[0152] Compositions are provided herein, wherein the composition comprises: (a) yeast cells, wherein the yeast cells comprise a permeable cell wall; and (b) one or more RNA adjuvants, wherein the one or more RNA adjuvants are used for at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. Compositions are provided herein, wherein the composition comprises: (a) yeast cells, wherein the yeast cells comprise a permeable cell wall; and (b) one or more nucleic acids, wherein the nucleic acids encode at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. Compositions are provided herein, wherein the composition comprises: (a) yeast cells, wherein the yeast cells comprise a permeable cell wall; and (b) one or more synthetic proteins, wherein the one or more synthetic proteins comprise at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. Compositions are also provided herein, wherein the composition further comprises RNA encoding a viral antigen. Compositions are also provided herein, wherein the composition further comprises DNA encoding a viral antigen. Compositions are also provided herein, wherein the composition further comprises chemically modified RNA encoding a viral antigen. Also provided herein are compositions wherein one or more RNA adjuvants or nucleic acids comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 175. Also provided herein are compositions wherein one or more RNA adjuvants or nucleic acids comprise a sequence having at least 90% identity to any one of SEQ ID NOs: 2 to 175. Also provided herein are compositions wherein one or more RNA adjuvants or nucleic acids comprise a sequence having at least 95% identity to any one of SEQ ID NOs: 2 to 175. Also provided herein are compositions wherein one or more RNA adjuvants or nucleic acids comprise any one of SEQ ID NOs: 2 to 175. Also provided herein are compositions wherein one or more RNA adjuvants or nucleic acids encode an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 176 to 217. Also provided herein are nucleic acids encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 206. Also provided herein are nucleic acids encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 210. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 212. Also provided herein are nucleic acids encoding an amino acid sequence having at least 85% identity to SEQ ID NO: 213. Also provided herein are nucleic acids encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 215.Also provided herein is a nucleic acid encoding an amino acid sequence having at least 75% identity to SEQ ID NO: 217. Also provided herein is a composition wherein one or more RNA adjuvants or nucleic acids are encapsulated within yeast cells. Also provided herein is a composition wherein the composition further comprises enteric coated microspheres wherein the yeast cells are encapsulated within the enteric coated microspheres. Also provided herein is a composition wherein the composition is formulated for oral administration to a subject.

[0153] Provided herein is a composition for inducing an immune response after being administered to a subject, wherein the composition comprises: (a) one or more carriers; (b) one or more nucleic acids, wherein one or more nucleic acid encoding: (i) at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist or any combination thereof; (ii) a tumor antigen; (iii) an immunomodulatory protein; or (iv) any combination of (i)-(iii); and (c) a delivery vehicle, wherein after administering an effective amount of the composition, in the presence of a tumor antigen, an innate immune response is induced in the subject, thereby inducing an immune response against a tumor antigen. Also provided herein is a composition, wherein one or more carriers include a lipid carrier. Also provided herein is a composition, wherein the immunomodulatory protein is CD83, a 4-1BB ligand or a cytokine. Also provided herein are compositions, wherein the tumor antigen is mucin 1 (MUC1), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (Her2 / neu), telomerase (TERT), survivin, melanoma-associated antigen A1 (MAGE-A1), MAGE-A3, Wilms tumor 1 (WT1), melanoma preferentially expressed antigen (PRAME), New York esophageal 1 (NY-ESO-1), tumor protein p53, tyrosinase, S100 protein, melanoma-associated antigen recognized by T cells 1 (MART-1), or any combination thereof. Also provided herein are compositions, wherein one or more nucleic acids comprise RNA, DNA, or a combination thereof. Also provided herein are compositions, wherein one or more nucleic acids encoding tumor antigens and / or immunomodulatory proteins are chemically modified. Also provided herein are compositions, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap. Also provided herein are compositions, wherein one or more lipid carriers are compounded with one or more RNA adjuvants. Also provided herein are compositions, wherein one or more lipid carriers are compounded with at least one RNA encoding tumor antigens and / or immunomodulatory proteins. Compositions are also provided herein, wherein one or more RNA adjuvants are encapsulated by one or more lipid carriers. Compositions are also provided herein, wherein at least one RNA encoding a tumor antigen and / or an immunomodulatory protein is encapsulated by one or more lipid carriers. Compositions are also provided herein, wherein one or more RNA adjuvants are operably linked to at least one RNA encoding a viral antigen, a derivative thereof, or a functional fragment thereof. Compositions are also provided herein, wherein one or more RNA adjuvants comprise a sequence selected from SEQ ID NO: 2 to 175, or a functional fragment thereof. Compositions are also provided herein, wherein the innate immune response induced in a subject is greater than the innate immune response in a subject to which an effective amount of the composition is not administered.

[0154] Provided herein are vectors, wherein the vector comprises: one or more nucleic acids encoding a TLR5 agonist, a TLR4 agonist, a TLR2 agonist, or a combination thereof. Provided herein are vectors, wherein the vector comprises a viral vector. Provided herein are vectors, wherein the viral vector comprises adenovirus, adeno-associated virus (AAV), recombinant AAV (rAAV), lentivirus, alphavirus, flavivirus, rhabdovirus, Kunjin virus, measles virus, Lassa virus, or its virus-like particles. Provided herein are vectors, wherein the TLR5 agonist comprises a functional derivative of a bacterial movement protein. Provided herein are vectors, wherein the TLR5 agonist comprises a DNA sequence encoding an RNA sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 37. Provided herein are vectors, wherein the TLR5 agonist comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 37. Also provided herein are vectors, wherein the TLR4 agonist comprises at least one functional fragment of a high mobility group protein B1 (HMGB1) protein, a nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, a transglycosylase, a 50S ribosomal protein, a heparin-binding hemagglutinin protein, a YadA family autotransporting adhesin, a dnaJ protein, a pneumolysin protein, an Omp19 protein, a 6,7-dimethyl-8-ribosylcytosine synthase, or any combination thereof. Also provided herein are vectors, wherein one or more nucleic acids comprise a DNA sequence encoding an RNA sequence having at least 85% identity to any one of SEQ ID NOs: 38 to 103. Also provided herein are vectors, wherein one or more nucleic acids comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 38 to 103. Also provided herein are vectors, wherein the TLR2 agonist comprises at least one functional fragment of an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system. Also provided herein are vectors, wherein one or more nucleic acids comprise a DNA sequence encoding an RNA sequence having at least 85% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are vectors, wherein one or more nucleic acids comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 104 to 175. Also provided herein are vectors, wherein the vectors further comprise a nucleic acid sequence encoding a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, or a tumor antigen. In some embodiments, the nucleic acid sequence is an RNA sequence or a DNA sequence. Also provided herein are vectors, wherein the viral antigen comprises a SARS-CoV-2 spike protein.

[0155] Provided herein are compositions, wherein the composition comprises a vector provided herein; and a lipid carrier.

[0156] Provided herein are vaccine compositions, wherein the vaccine compositions comprise a composition provided herein; and a pharmaceutically acceptable excipient.

[0157] Provided herein are vaccine compositions, wherein the vaccine compositions comprise: (a) a lipid carrier; (b) one or more RNA adjuvants, wherein the one or more RNA adjuvants comprise a sequence having at least 85% identity to any one of SEQ ID NOs: 2 to 175; and (c) one or more RNAs encoding an antigen. Also provided herein are vaccine compositions, wherein the one or more RNAs encoding an antigen each encode a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or any combination thereof.

[0158] Provided herein is a method for stimulating an immune response of a subject, the method comprising: administering to the subject an effective amount of a composition provided herein or a vaccine composition provided herein, thereby stimulating an immune response. Also provided herein is a method for stimulating an immune response of a subject, wherein administration is systemic administration or local administration. Also provided herein is a method for stimulating an immune response of a subject, wherein administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumoral administration, inhalation, subcutaneous administration, or intraperitoneal administration. Also provided herein is a method for stimulating an immune response of a subject, wherein administration is daily, weekly, monthly, or annually. Also provided herein is a method for stimulating an immune response of a subject, wherein the method further comprises administering an RNA vaccine composition to the subject. Also provided herein is a method for stimulating an immune response of a subject, wherein the subject suffers from an infection or is at risk of developing an infection. Also provided herein is a method for stimulating an immune response of a subject, wherein the infection is a viral infection. Also provided herein is a method for stimulating an immune response of a subject, wherein the viral infection is an upper respiratory tract viral infection. Also provided herein is a method for stimulating an immune response in a subject, wherein the upper respiratory tract viral infection is COVID-19, SARS infection, MERS infection, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia or rhinovirus infection. Also provided herein is a method for stimulating an immune response in a subject, wherein the infection is a bacterial infection, a fungal infection, a parasitic infection or a yeast infection. Also provided herein is a method for stimulating an immune response in a subject, wherein the antigen is a spike protein, a glycoprotein, a viral envelope protein or a tumor antigen. Also provided herein is a method for stimulating an immune response in a subject, wherein the spike protein is from a SARS-CoV-2 virus. Also provided herein is a method for stimulating an immune response in a subject, wherein the subject is a mammal. Also provided herein is a method for stimulating an immune response in a subject, wherein the subject is a human. Also provided herein is a method for stimulating an immune response in a subject, wherein an additional dose of a composition or a vaccine composition is administered to the subject.

[0159] Provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, the method comprising: (a) administering an effective amount of a composition provided herein or a vaccine composition provided herein to a subject; and (b) administering an RNA vaccine composition to a subject, wherein the RNA vaccine composition comprises RNA encoding an antigen, thereby enhancing an immune response to the antigen encoded by the RNA. Also provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein administration is systemic administration or local administration. Also provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumoral administration, inhalation, subcutaneous administration, or intraperitoneal administration. Also provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein administration is daily, weekly, monthly, or annual. Also provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the subject suffers from an infection or is at risk of developing an infection. Also provided herein is a method for enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the infection is a viral infection. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the viral infection is an upper respiratory tract viral infection. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the upper respiratory tract viral infection is COVID-19, SARS infection, MERS infection, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia or rhinovirus infection. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the infection is a bacterial infection, a fungal infection, a parasitic infection or a yeast infection. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the spike protein is from a SARS-CoV-2 virus. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the subject is a mammal. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein the subject is a human. Also provided herein is a method for enhancing the immune response to an antigen encoded by RNA in an RNA vaccine composition, wherein an additional dose of a composition or vaccine composition is administered to the subject. Also provided herein are methods of enhancing an immune response to an antigen encoded by the RNA in an RNA vaccine composition, wherein the subject has cancer, has been diagnosed with cancer, or is at risk of developing cancer. Also provided herein are methods of enhancing an immune response to an antigen encoded by the RNA in an RNA vaccine composition, wherein the subject has a solid tumor or a blood cancer.Also provided herein are methods of enhancing an immune response to an antigen encoded by the RNA in an RNA vaccine composition, wherein the cancer is lung cancer, breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, lung cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer, head and neck cancer, or thyroid cancer.

[0160] Provided herein is a method for treating an infection in a subject, the method comprising: administering to the subject an effective amount of a composition provided herein or a vaccine composition provided herein, thereby treating the infection in the subject. Also provided herein is a method for treating an infection in a subject, wherein administration is systemic administration or topical administration. Also provided herein is a method for treating an infection in a subject, wherein administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumor administration, inhalation, subcutaneous administration, or intraperitoneal administration. Also provided herein is a method for treating an infection in a subject, wherein administration is daily, weekly, monthly, or annually. In some embodiments, administration is at least about every 6 hours, 12 hours, 24 hours (1 day), 48 hours (2 days), 72 hours (3 days), 96 hours (4 days), 120 hours (5 days), 144 hours (6 days), 168 hours (7 days), 240 hours (10 days), 336 hours (14 days), 504 hours (21 days), 672 hours (28 days), 720 hours (30 days, one month), 840 hours (35 days), 1344 hours (56 days), 8760 hours (365 days, 1 year) or longer. Also provided herein is a method for treating an infection in a subject, wherein the method further comprises administering an RNA vaccine composition to the subject, wherein the infection is a viral infection. Also provided herein is a method for treating an infection in a subject, wherein the viral infection is an upper respiratory tract viral infection. Also provided herein is a method for treating an infection in a subject, wherein the infection is a tumor virus infection. Also provided herein is a method for treating an infection in a subject, wherein an upper respiratory tract viral infection is COVID-19, SARS infection, MERS infection, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia or rhinovirus infection. Also provided herein is a method for treating an infection in a subject, wherein the infection is a bacterial infection, a fungal infection, a parasitic infection or a yeast infection. Also provided herein is a method for treating an infection in a subject, wherein the antigen is a spike protein, a glycoprotein, a viral envelope protein or a tumor antigen. Also provided herein is a method for treating an infection in a subject, wherein the spike protein is from a SARS-CoV-2 virus. Also provided herein is a method for treating an infection in a subject, wherein the tumor virus infection is from a human papillomavirus (HPV), a Kaposi's sarcoma-associated herpesvirus (KSHV), a Merkel cell polyomavirus (MCV), a human T-cell lymphotropic virus type 1 (HTLV-1) or an Epstein-Barr virus (EBV). Also provided herein is a method for treating an infection in a subject, wherein the subject is a mammal. Also provided herein is a method for treating an infection in a subject, wherein the subject is a human. Also provided herein are methods for treating an infection in a subject, wherein an additional dose of the composition or vaccine composition is administered to the subject.

[0161] Provided herein is a method for treating a subject's cancer, the method comprising: administering to the subject an effective amount of a composition provided herein or a vaccine composition provided herein, thereby treating the subject's cancer. Also provided herein is a method for treating cancer, wherein administration is systemic administration or local administration. Also provided herein is a method for treating cancer, wherein administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumoral administration, inhalation, subcutaneous administration, or intraperitoneal administration. Also provided herein is a method for treating cancer, wherein administration is daily, weekly, monthly, or annually. In some embodiments, administration is at least about every 6 hours, 12 hours, 24 hours (1 day), 48 hours (2 days), 72 hours (3 days), 96 hours (4 days), 120 hours (5 days), 144 hours (6 days), 168 hours (7 days), 240 hours (10 days), 336 hours (14 days), 504 hours (21 days), 672 hours (28 days), 720 hours (30 days, one month), 840 hours (35 days), 1344 hours (56 days) or 8760 hours (365 days, 1 year). Also provided herein is a method for treating cancer, wherein the method further comprises administering an RNA vaccine composition to a subject. Also provided herein is a method for treating cancer, wherein the subject suffers from cancer, is diagnosed with cancer, or is at risk of developing cancer. Also provided herein is a method for treating cancer, wherein the subject suffers from a solid tumor or a blood cancer. Also provided herein is a method for treating cancer, wherein the cancer is lung cancer, breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, lung cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer, head and neck cancer or thyroid cancer. Also provided herein is a method for treating cancer, wherein the composition comprises mRNA encoding a tumor antigen. Also provided herein is a method for treating cancer, wherein the tumor antigen is mucin 1 (MUC1), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (Her2 / neu), telomerase (TERT), survivin, melanoma-associated antigen A1 (MAGE-A1), MAGE-A3, Wilms tumor 1 (WT1), melanoma preferentially expressed antigen (PRAME), New York esophagus 1 (NY-ESO-1), tumor protein p53, tyrosinase, S100 protein, melanoma-associated antigen recognized by T cells 1 (MART-1) or any combination thereof.

[0162] Although preferred embodiments of the present invention 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, changes and substitutions will occur to those skilled in the art without departing from the present invention.

[0163] Example

[0164] Example 1: Mechanisms by which RNA adjuvants induce innate and adaptive immune responses.

[0165] The functional mechanism of RNA adjuvants is described below.

[0166] Figure 1 A schematic diagram of the improved mRNA adjuvant approach for mRNA vaccines is shown. Figure 1 (Top section) provides a mechanism for immune stimulation by conventional mRNA vaccines, wherein lipid nanoparticles or carriers of RNA encoding antigens are internalized by target cells and recognized by cell signaling mechanisms (such as TLR7 / 8 and RIG-1), which leads to downstream type 1 interferon responses (IFN). For conventional mRNA vaccines, mRNA is transported into host cells by lipid nanoparticles. The host cell translation mechanism translates mRNA into antigenic proteins, which are then presented by cells together with major histocompatibility complex (MHC) molecules. Class II MHC molecules present antigens to CD4+ helper T cells. Class I MHC molecules present antigens to CD8+ cytotoxic T cells. After the presented antigens and MHC class II molecules interact with CD4+ helper T cell receptors, CD4+ lymphocytes are activated, IL-2 is released, and IL-2 receptors are expressed on the surface of CD4+ lymphocytes. IL-2 produced by activated cells stimulates its own receptors, as well as the receptors of mononuclear phagocytes, thereby increasing its microbicidal activity. IL-2 also stimulates B cells to synthesize antibodies. While B cells can recognize protein antigens in their native state, T cells only recognize peptides produced during antigen processing, i.e., major histocompatibility complex molecules. Traditional vaccines can activate the production of interferon (IFN), which can block effective translation and reduce antigen production in host cells. IFN signaling induces a Th-1 polarized response.

[0167] Figure 1 (Bottom) provides the mRNA Flag Mechanism of adjuvant-powered mRNA vaccines, which leads to the induction of NF-kB signaling through TLR5, TLR4 or TLR2 stimulation, and the release of immunostimulatory cytokines and chemokines is expected to complement and act in a synergistic manner with the mRNA-mediated IFN response to mobilize the immune response to the antigen production site encoded by the mRNA vaccine. Since TLR5 agonistic adjuvants achieve a balanced Th1- / Th2-polarized immune response, it leads to an improvement in the quality of the immune response.

[0168] Example 2: RNA adjuvant production, purification and characterization.

[0169] The production of RNA adjuvants and RNA-encoded antigens is described. The RNA is synthesized by in vitro transcription of a DNA fragment encoding all the elements. The cap structure can be covalently attached to the 5'UTR by different capping enzymes in a co-transcriptional or post-transcriptional manner.

[0170] The final RNA product is purified using affinity chromatography on oligo-dT to remove impurities generated during transcription (such as double-stranded RNA), which can effectively activate the innate immune response.

[0171] RNA can be additionally modified. The untranslated region (UTR) of RNA is optimized using machine learning techniques trained according to the ribosome loading spectrum of the reporter gene library, wherein the 5'UTR contains a completely random sequence. This is further tested and verified in the library of human UTR and variants associated with human diseases. Reporter genes can also be used and connected to random cDNA obtained from the fragmented mRNA isolated from human dendritic cells by reverse transcription at the 3' end for 3'UTR modification. Adding the second 3'UTR from different genes can further enhance mRNA stability and protein expression. In addition, by testing the level of reporter gene protein expressed from mRNA of different poly A tail lengths, RNA can be optimized with poly A tails. Modified nucleobases can be used to further stabilize RNA adjuvants and RNA encoding antigens as described herein.

[0172] Characterization of the RNA can be accomplished using a method selected from polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, and RNA impurity detection, wherein the characterization comprises determining the sequence of the RNA transcript, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript.

[0173] Example 3: RNA synthesis and purification.

[0174] The mRNA compositions provided herein can be synthesized using T7 phage. In short, mRNA is codon optimized, synthesized and cloned into mRNA production plasmid. After being connected to the expression vector, T7 RNA polymerase is used to produce mRNA on the linear plasmid. The purification of mRNA is carried out by dialysis or spin column chromatography based on beads.

[0175] Example 4: Complexation of RNA with lipid carriers.

[0176] The RNA adjuvant and mRNA encoding the SAR-CoV-2 spike protein are compounded with a lipid carrier. The lipid carrier can be composed of ionizable cationic lipids, cholesterol, phospholipids (such as distearoylphosphatidylcholine) and polyethylene glycol (PEG)-lipids. The spike protein can include a signal sequence, an S1 domain and an S2 domain that target the antigen to the endoplasmic reticulum of the cell, and a receptor binding domain (RBD), followed by a short transmembrane domain and a cytoplasmic domain. The RNA can further encode a furin cleavage site that stabilizes the prefusion conformation of the spike protein.

[0177] Table 2 and the sequences section herein provide RNA adjuvant sequences. The RNA can be mixed with a desired lipid carrier to form a vaccine composition. The resulting configuration of the RNA adjuvant with RNA encoding the spike is provided in FIG. 2A to FIG. 2D middle.

[0178] Configuration 1: RNA adjuvant and RNA encoding antigen are encapsulated in the same lipid carrier ( Figure 2A ).

[0179] Configuration 2: RNA adjuvant and RNA encoding antigen are encapsulated in different lipid carriers ( Figure 2B ).

[0180] Configuration 3: RNA adjuvant and RNA encoding antigen are on the same nucleic acid strand and encapsulated in a lipid carrier ( Figure 2C ).

[0181] Configuration 4: The RNA is bicistronic. In this configuration, the RNA sequence encoding the adjuvant is translated from the internal ribosome entry site (IRES), or the RNA adjuvant is separated from the antigen coding region of the RNA by a stop codon, which results in a relative reduction in the translation of the downstream open reading frame ( Figure 2D ).

[0182] Example 5: TLR5 agonist adjuvant compositions and NF-κB signaling in vitro.

[0183] mRNA (SEQ ID NO: 2, SEQ ID NO: 176) encoding TLR5 agonist adjuvant (TLR5 agonist 1) was produced by in vitro transcription (IVT). mRNA was produced by transcribing a linearized plasmid DNA template (e.g., SEQ ID NO: 226). Escherichia coli containing the plasmid was amplified in a bioreactor by fermentation and then harvested to produce a purified plasmid. As described in Examples 2 and 3 above, mRNA was produced from the purified plasmid by T7 RNA polymerase.

[0184] HEK293 cells (hereinafter referred to as HEK293-NFkB-Luc cells) were infected with a lentivirus containing a luciferase reporter gene driven by NF-κB to generate a stable cell line. When toll-like receptors (TLRs) were activated by agonists, the luciferase activity in HEK293-NFkB-Luc cells increased, as measured by a luminescence detector. In addition, when TLRs were activated by mRNA adjuvants, the amount of secreted proteins encoded by the mRNA adjuvants also increased, as measured by enzyme-linked immunosorbent assays (ELISA).

[0185] To determine the effect of mRNA adjuvants on TLR5-induced NF-κB signaling in vitro, HEK293-NFkB-Luc cells were transfected with TLR5 agonist 1 using polyethyleneimine (PEI) and compared with control HEK293-NFkB-Luc cells (null) ( Figure 3A ). Luciferase activity readings were performed and measured at 0 hours, 18 hours, and 40 hours after transfection with mRNA. NF-κB was activated by TLR5 agonist 1 ( Figure 3B Similarly, at 18 and 40 hours post-transfection, protein secretion of TLR5 agonists was increased in cells transfected with TLR5 agonist 1 relative to control cells ( Figure 3C ).

[0186] Next, TLR5 agonist 1 mRNA was complexed with lipid carriers to determine if lipid nanoparticle packaging improves mRNA delivery.Lipid nanoparticles containing a cationic lipid surface including PEG and DSPC as well as cholesterol were generated and purified.

[0187] Purified LNPs complexed with TLR5 agonist 1 were delivered to HEK293-NFkB-Luc cells. The time dependence of luciferase reporter activity was measured using HEK293-NFkB-Luc cells before (0 h) and 1 h after incubation with TLR5 agonist 1 protein (500 ng / ml), LNP-encapsulated RNA TLR5 agonist 1 (LNP (TLR5 agonist 1 RNA), 45 ng / ml) or LNP control (LNP (GFP)). For each condition, luciferase activity was measured every hour after transfection until 49 h ( Figure 4A). Culture media was collected from each treated cell line for ELISA analysis of TLR5 protein secretion. LNPs without mRNA adjuvant (LNP(GFP)) did not activate the NF-κB responsive luciferase reporter gene. In contrast, cell lines treated with LNP-encapsulated mRNA TLR5 agonist (LNP(TLR5 agonist 1 RNA)) or TLR5 agonist 1 protein showed increased luciferase activity relative to the control. This result indicates that the observed effect is specific to the TLR5 agonist adjuvant. These results also show that LNP delivery of RNA encoding TLR5 agonists can cause extensive secretion of TLR5 agonist proteins relative to delivery of protein alone or LNP controls, thereby enhancing TLR5 / NFκB cell signaling ( Figure 4B ).

[0188] LNP-encapsulated TLR5 agonist 1 RNA was administered to HEK293-NFkB-Luc cells expressing human TLR5 or HEK293-NFkB-Luc cells not expressing human TLR5 ( FIG. 5A to FIG. 5B ). LNP (TLR5 agonist 1 RNA) increased TLR5 luciferase activity in cells expressing human TLR5 relative to cells not expressing human TLR5 ( Figure 5A ) and TLR5 agonist 1 protein secretion ( Figure 5B ).

[0189] Example 6: In vivo biodistribution of LNP-mRNA adjuvants.

[0190] A mouse model was used to test the in vivo biodistribution of RNA delivered by lipid nanoparticles. 38-week-old BALB / c-Tg(Rela-luc)31Xen females were injected intramuscularly (IM) with 20 μl of LNP-TLR5 agonist 1 (100 ng / μl of RNA), LNP-GFP, or PBS ( Figure 6 ). After injection of D-firefly luciferase potassium salt (Gold Bio), animals were imaged for 5 minutes using a Perkin Elmer IVIS Spectrum in vivo imager under anesthesia. Total luminescence was measured for animals treated with LNP-TLR5 agonist 1; animals treated with LNP-GFP; and animals treated with PBS at 0, 3, 5, 6, 8, 10, 12, 15, 18, and 36 hours post-injection. Animals treated with LNP-TLR5 agonist mRNA showed high levels of D-firefly luciferin, and TLR5 protein was widely distributed throughout the animal ( Figure 7 ). The production and secretion of TLR5 agonists and TLR5-dependent NF-kB activation were confirmed in vitro and in vivo. No adverse events were detected in the treated animals.

[0191] It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are therefore covered.

Claims

1. A composition for inducing an immune response after administration to a subject, the composition comprising: (a) an RNA adjuvant encoding a synthetic protein, wherein the synthetic protein comprises at least one functional fragment of a toll-like receptor 5 (TLR5) agonist, which is capable of activating the TLR5 pathway in a cell when in contact with the cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, Wherein, upon administration of an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject.

2. The composition of claim 1, wherein at least one functional fragment of the TLR5 agonist comprises a functional derivative of a bacterial movement protein.

3. The composition of claim 2, wherein the bacterial movement protein is bacterial flagellin, a derivative or a functional fragment thereof.

4. The composition of claim 1, wherein the synthetic protein is a truncated form of bacterial flagellin, a secreted form of bacterial flagellin, or a combination thereof.

5. The composition of claim 1, wherein the functional fragment of the toll-like receptor 5 (TLR5) agonist comprises the D0 region and the D1 region of the bacterial flagellin.

6. The composition according to any one of claims 1 to 5, wherein the functional fragment of the toll-like receptor 5 (TLR5) agonist comprises the D0 region, the D1 region and the D2 region of the bacterial flagellin.

7. The composition of claim 1, wherein the functional fragment of the toll-like receptor 5 (TLR5) agonist comprises the D0 region, the D1 region, the D2 region and the D3 region of the bacterial flagellin.

8. The composition of claim 1, wherein the synthetic protein further comprises a signal sequence.

9. The composition of claim 8, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO:

1.

10. The composition of claim 1, wherein the RNA comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 2 to 37.

11. The composition of claim 1, wherein the RNA comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 2 to 37.

12. The composition of claim 1, wherein the RNA comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 to 37.

13. The composition of claim 1, wherein the RNA comprises a sequence selected from any one of SEQ ID NOs: 2 to 37.

14. The composition of claim 1, wherein the one or more delivery vehicles are diluents, aqueous solutions, particles, emulsions, suspensions, lipid carriers, vesicles, carriers, or any combination thereof.

15. The composition of claim 1, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen.

16. The composition of claim 1, wherein the antigen is a tumor antigen.

17. The composition of claim 1, further comprising RNA encoding the antigen, wherein the RNA encoding the antigen and the RNA adjuvant are in the same delivery vehicle.

18. The composition of claim 1, further comprising RNA encoding the antigen, wherein the RNA encoding the antigen and the RNA adjuvant are in different delivery vehicles.

19. The composition of claim 1, wherein the RNA adjuvant further comprises an RNA sequence encoding the antigen.

20. The composition of claim 19, wherein the RNA sequence encoding the antigen is operably linked to the RNA adjuvant.

21. The composition of claim 1, wherein the innate immune response comprises an increase in the level or activity of nuclear factor kappa B (NF-κB) in epithelial cells of the subject relative to the level or activity of NF-κB in the absence of the composition.

22. A composition comprising: (a) a lipid carrier; and (b) an RNA sequence encoding at least a functional fragment of a bacterial movement protein, wherein the functional fragment of the bacterial movement protein is a TLR5 agonist.

23. The composition of claim 22, wherein the RNA sequence is encapsulated within the lipid carrier.

24. The composition of claim 22, wherein the RNA sequence is complexed with the lipid carrier.

25. The composition of claim 22, wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipid complex, a lipid class, a derivative, a variant, or any combination thereof.

26. The composition of claim 22, wherein the at least one functional fragment of the bacterial movement protein is a bacterial flagellin, a derivative or a functional fragment thereof.

27. The composition of claim 22, wherein the bacterial movement protein is a truncated form of bacterial flagellin, a secreted form of bacterial flagellin, or a combination thereof.

28. The composition of claim 22, wherein the functional fragment of the bacterial movement protein comprises the D0 region and the D1 region of the bacterial flagellin.

29. The composition of claim 22, wherein the functional fragment of the bacterial movement protein comprises the D0 region, the D1 region, and the D2 region of the bacterial flagellin.

30. The composition of claim 22, wherein the functional fragment of the bacterial movement protein comprises the D0 region, the D1 region, the D2 region, and the D3 region of the bacterial flagellin.

31. The composition of claim 22, wherein the RNA sequence comprises mRNA.

32. A composition as described in claim 22, wherein the composition further comprises one or more RNA sequences each encoding an antigen.

33. The composition of claim 22, wherein the composition further comprises one or more antigens.

34. The composition of claim 32, wherein the RNA encoding the antigen is mRNA.

35. The composition of claim 34, wherein the mRNA is chemically modified.

36. The composition of claim 35, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap.

37. The composition of claim 32, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

38. The composition of claim 37, wherein the viral antigen is from an adenovirus, an astrovirus, a bocavirus, a Bannavirus, a BK virus, a Coltivirus, a coronavirus, a coxsackievirus, a cytomegalovirus, a dengue virus, an Ebola virus, an enterovirus, an Epstein-Barr virus, a hantavirus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus, a human immunodeficiency virus (HIV), an influenza virus, a JC virus, a Lassa virus, a Marburg virus, a measles virus, a Middle East respiratory syndrome (MERS) coronavirus, a Norwalk virus, a Norovirus, a Parvovirus, a Papillomavirus, a Poliovirus, a Rabies virus, a Rhinovirus, a Rotavirus, a Rubella virus, a Severe Acute Respiratory Syndrome (SARS) virus, a Smallpox virus, a Varicella-Zoster virus, a Yellow Fever virus, or any combination thereof.

39. The composition of claim 38, wherein the SARS virus is a SARS-CoV-2 virus.

40. The composition of claim 32, wherein the antigen comprises a spike protein, a glycoprotein, or a hemagglutinin protein.

41. The composition of claim 32, wherein the RNA encoding the antigen and the RNA adjuvant are encapsulated in the same lipid carrier.

42. The composition of claim 32, wherein the RNA encoding the antigen and the RNA adjuvant are encapsulated in different lipid carriers.

43. The composition of claim 22, wherein the RNA adjuvant comprises a sequence at least 85% identical to any one of SEQ ID NOs: 2 to 37.

44. The composition of claim 22, wherein the RNA adjuvant comprises a sequence at least 90% identical to any one of SEQ ID NOs: 2 to 37.

45. The composition of claim 22, wherein the RNA adjuvant comprises a sequence at least 95% identical to any one of SEQ ID NOs: 2 to 37.

46. ​​The composition of claim 22, wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NOs: 2 to 37.

47. The composition of claim 22, wherein the composition is in the form of a suspension, an aqueous solution, or an emulsion.

48. A composition for inducing an immune response when administered to a subject, the composition comprising: (a) an RNA adjuvant encoding a synthetic protein, wherein the synthetic protein comprises at least one functional fragment of a toll-like receptor 4 (TLR4) agonist, wherein the functional fragment is capable of activating a TLR4 pathway in a cell when in contact with the cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein upon administration of an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject, thereby inducing an immune response in the presence of the antigen.

49. The composition of claim 48, wherein the TLR4 agonist comprises at least one functional fragment of a high mobility group protein B1 (HMGB1) protein, a nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, a transglycosylase, a 50S ribosomal protein, a heparin-binding hemagglutinin protein, a YadA family autotransporting adhesin, a dnaJ protein, a pneumolysin protein, an Omp19 protein, a 6,7-dimethyl-8-ribosylcytosine synthase, or any combination thereof.

50. The composition of claim 48, wherein the TLR4 agonist is from bacteria.

51. The composition of claim 48, wherein the TLR4 agonist is from a human.

52. The composition of claim 48, wherein the RNA adjuvant comprises a sequence at least 85% identical to any one of SEQ ID NOs: 38 to 103.

53. The composition of claim 48, wherein the RNA adjuvant comprises a sequence at least 90% identical to any one of SEQ ID NOs: 38 to 103.

54. The composition of claim 48, wherein the RNA adjuvant comprises a sequence at least 95% identical to any one of SEQ ID NOs: 38 to 103.

55. The composition of claim 48, wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NOs: 38 to 103.

56. The composition of claim 48, wherein the delivery vehicle is a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, or any combination thereof.

57. The composition of claim 48, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

58. The composition of claim 48, wherein the innate immune response comprises an increase in the level or activity of NF-κB when the composition is administered relative to the level or activity of NF-κB in the absence of the composition.

59. A composition comprising: (a) a lipid carrier; and (b) one or more RNA sequences, wherein the one or more RNA sequences encode at least a functional fragment of a TLR4 agonist, wherein the functional fragment of the TLR4 agonist is capable of activating a TLR4 pathway in a cell when in contact with the cell.

60. The composition of claim 59, wherein the TLR4 agonist comprises at least one functional fragment of a high mobility group protein B1 (HMGB1) protein, a nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, a transglycosylase, a 50S ribosomal protein, a heparin-binding hemagglutinin protein, a YadA family autotransporting adhesin, a dnaJ protein, a pneumolysin protein, an Omp19 protein, a 6,7-dimethyl-8-ribosylcytosine synthase, or any combination thereof.

61. The composition of claim 59, wherein the TLR4 agonist is from bacteria.

62. The composition of claim 59, wherein the TLR4 agonist is from a human.

63. The composition of claim 59, wherein the RNA sequence comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 38 to 103.

64. The composition of claim 59, wherein the RNA sequence comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 38 to 103.

65. The composition of claim 59, wherein the RNA sequence comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 38 to 103.

66. The composition of claim 59, wherein the RNA sequence comprises a sequence selected from any one of SEQ ID NOs: 38 to 103.

67. The composition of claim 59, wherein the RNA sequence is encapsulated within the lipid carrier.

68. The composition of claim 59, wherein the RNA sequence is complexed with the lipid carrier.

69. The composition of claim 59, wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipid complex, a lipid class, a derivative, a variant, or any combination thereof.

70. The composition of claim 59, wherein the RNA adjuvant comprises mRNA.

71. A composition as described in claim 59, wherein the composition also comprises an RNA sequence encoding an antigen.

72. The composition of claim 59, wherein the composition further comprises one or more antigens.

73. The composition of claim 71, wherein the RNA sequence encoding the antigen is mRNA.

74. The composition of claim 73, wherein the mRNA is chemically modified.

75. The composition of claim 74, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap.

76. The composition of claim 71, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

77. The composition of claim 76, wherein the viral antigen is from an adenovirus, an astrovirus, a bocavirus, a Bannavirus, a BK virus, a Coltivirus, a coronavirus, a coxsackievirus, a cytomegalovirus, a dengue virus, an Ebola virus, an enterovirus, an Epstein-Barr virus, a hantavirus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus, a human immunodeficiency virus (HIV), an influenza virus, a JC virus, a Lassa virus, a Marburg virus, a measles virus, a Middle East respiratory syndrome (MERS) coronavirus, a Norwalk virus, a Norovirus, a Parvovirus, a Papillomavirus, a Poliovirus, a Rabies virus, a Rhinovirus, a Rotavirus, a Rubella virus, a Severe Acute Respiratory Syndrome (SARS) virus, a Smallpox virus, a Varicella-Zoster virus, a Yellow Fever virus, or any combination thereof.

78. The composition of claim 77, wherein the SARS virus is a SARS-CoV-2 virus.

79. The composition of claim 71, wherein the antigen comprises a spike protein, a glycoprotein, or a hemagglutinin protein.

80. The composition of claim 71, wherein the RNA encoding the antigen and the RNA sequence encoding the TLR4 agonist are encapsulated in the same lipid carrier.

81. The composition of any one of claims 71 to 80, wherein the RNA encoding the antigen and the one or more RNA sequences encoding at least the functional fragment of the TLR4 agonist are encapsulated in different lipid carriers.

82. The composition of claim 59, wherein the composition is in the form of a suspension, an aqueous solution or an emulsion.

83. A composition for inducing an immune response when administered to a subject, the composition comprising: (a) an RNA adjuvant encoding a synthetic protein, wherein the synthetic protein comprises at least one functional fragment of a toll-like receptor 2 (TLR2) agonist, wherein the functional fragment is capable of activating the TLR2 pathway in a cell when in contact with the cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein upon administration of an effective amount of the composition to a subject in the presence of an antigen, an initial innate immune response is induced in the subject, thereby inducing an immune response in the presence of the antigen.

84. The composition of claim 83, wherein the TLR2 agonist comprises at least one functional fragment of an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system.

85. The composition of claim 83, wherein the TLR2 agonist is from bacteria.

86. The composition of claim 83, wherein the RNA adjuvant comprises a sequence at least 85% identical to any one of SEQ ID NOs: 104 to 175.

87. The composition of claim 83, wherein the RNA adjuvant comprises a sequence at least 90% identical to any one of SEQ ID NOs: 104 to 175.

88. The composition of claim 83, wherein the RNA adjuvant comprises a sequence at least 95% identical to any one of SEQ ID NOs: 104 to 175.

89. The composition of claim 83, wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NOs: 104 to 175.

90. The composition of claim 83, wherein the delivery vehicle is a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, or any combination thereof.

91. The composition of claim 83, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

92. The composition of claim 83, wherein the innate immune response comprises an increase in the level or activity of NF-κB when the composition is administered relative to the level or activity of NF-κB in the absence of the composition.

93. A composition comprising: (a) a lipid carrier; and (b) one or more RNA sequences, wherein the one or more RNA sequences encode at least a functional fragment of a TLR2 agonist, wherein the functional fragment of the TLR2 agonist activates a TLR2 pathway in a cell when in contact with the cell.

94. The composition of claim 93, wherein the TLR2 agonist comprises at least one functional fragment of an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system.

95. The composition of claim 93, wherein the TLR2 agonist is from bacteria.

96. The composition of claim 93, wherein the one or more RNA sequences comprise a sequence that is at least 85% identical to any one of SEQ ID NOs: 104 to 175.

97. The composition of claim 93, wherein the one or more RNA sequences comprise a sequence that is at least 90% identical to any one of SEQ ID NOs: 104 to 175.

98. The composition of claim 93, wherein the one or more RNA sequences comprise a sequence that is at least 95% identical to any one of SEQ ID NOs: 104 to 175.

99. The composition of claim 93, wherein the one or more RNA sequences comprise a sequence selected from any one of SEQ ID NOs: 104 to 175.

100. The composition of claim 93, wherein the one or more RNA sequences are encapsulated within the lipid carrier.

101. The composition of claim 93, wherein the one or more RNA sequences are complexed with the lipid carrier.

102. The composition of claim 93, wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymer-conjugated lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipid complex, a lipid class, a derivative, a variant, or any combination thereof.

103. The composition of claim 93, wherein the one or more RNA sequences comprise mRNA.

104. The composition of claim 93, wherein the composition further comprises an RNA sequence encoding an antigen.

105. The composition of claim 93, wherein the composition further comprises one or more antigens.

106. The composition of claim 104, wherein the RNA encoding the antigen is mRNA.

107. The composition of claim 106, wherein the mRNA is chemically modified.

108. The composition of claim 107, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap.

109. The composition of claim 104, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

110. The composition of claim 109, wherein the viral antigen is from an adenovirus, an astrovirus, a bocavirus, a Bannavirus, a BK virus, a Coltivirus, a coronavirus, a coxsackievirus, a cytomegalovirus, a dengue virus, an Ebola virus, an enterovirus, an Epstein-Barr virus, a hantavirus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus, a human immunodeficiency virus (HIV), an influenza virus, a JC virus, a Lassa virus, a Marburg virus, a measles virus, a Middle East respiratory syndrome (MERS) coronavirus, a Norwalk virus, a Norovirus, a Parvovirus, a Papillomavirus, a Poliovirus, a Rabies virus, a Rhinovirus, a Rotavirus, a Rubella virus, a Severe Acute Respiratory Syndrome (SARS) virus, a Smallpox virus, a Varicella-Zoster virus, a Yellow Fever virus, or any combination thereof.

111. The composition of claim 110, wherein the SARS virus is a SARS-CoV-2 virus.

112. The composition of claim 104, wherein the antigen comprises a spike protein, a glycoprotein, a hemagglutinin protein, or a cancer cell protein.

113. The composition of claim 104, wherein the RNA sequence encoding the antigen and the RNA adjuvant are encapsulated in the same lipid carrier.

114. The composition of claim 104, wherein the RNA sequence encoding the antigen and the one or more RNA sequences encoding the functional fragment of the TLR2 agonist are encapsulated in different lipid carriers.

115. The composition of claim 93, wherein the composition is in the form of a suspension, an aqueous solution, or an emulsion.

116. A composition for inducing an immune response when administered to a subject, comprising: (a) one or more carriers; (b) one or more nucleic acids, wherein the one or more nucleic acids encode: (i) at least one functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or any combination thereof, wherein the functional fragment activates a TLR4 pathway, a TLR2 pathway, or a TLR5 pathway in a cell when administered to a subject; (ii) viral antigens; or (iii) a combination of (i) and (ii); and (c) a delivery vehicle, wherein upon administration of an effective amount of the composition, an innate immune response is induced in the subject in the presence of a viral antigen, thereby inducing an immune response against the viral antigen.

117. The composition of claim 116, wherein the viral antigen is a spike protein, a glycoprotein, or a viral envelope protein.

118. The composition of claim 117, wherein the spike protein is from a SARS-CoV-2 virus.

119. The composition of claim 116, wherein the one or more nucleic acids encoding the viral antigens are mRNAs.

120. The composition of claim 116, wherein the one or more nucleic acids encoding the viral antigens are chemically modified.

121. The composition of claim 120, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap.

122. The composition of claim 116, wherein the one or more carriers are complexed with the one or more nucleic acids.

123. The composition of claim 116, wherein the one or more carriers are complexed with one or more nucleic acid sequences encoding the viral antigens.

124. The composition of claim 116, wherein the one or more nucleic acids are encapsulated by the one or more carriers.

125. The composition of claim 116, wherein the one or more nucleic acids encoding the viral antigens are encapsulated by the one or more carriers.

126. The composition of claim 116, wherein the one or more nucleic acids are operably linked to at least one nucleic acid encoding the viral antigen.

127. The composition of claim 116, wherein the nucleic acid comprises a sequence selected from SEQ ID NOs: 2 to 175 or a functional fragment thereof.

128. The composition of claim 116, wherein the innate immune response induced in the subject is greater than the innate immune response in a subject to which an effective amount of the composition is not administered.

129. The composition of claim 116, wherein the innate immune response comprises an increase in the level or activity of NF-κB when the effective amount of the composition is administered relative to the level or activity of NF-κB in the absence of the composition.

130. A composition for inducing an immune response when administered to a subject, comprising: (a) one or more carriers; (b) one or more nucleic acids, wherein the one or more nucleic acids encode: (i) at least one functional fragment of a TLR4 agonist, a TLR2 agonist or a TLR5 agonist or any combination thereof, wherein the functional fragment activates a TLR4 pathway, a TLR2 pathway or a TLR5 pathway in a cell when administered to a subject; (ii) tumor antigens; or (iii) a combination of (i) or (ii); and (c) a delivery vehicle, Wherein, after administration of an effective amount of the composition, an innate immune response is induced in the subject in the presence of a tumor antigen, thereby inducing an immune response against the tumor antigen.

131. compositions as described in claim 130, wherein said composition also comprises the nucleic acid sequence encoding immunomodulatory protein, wherein said immunomodulatory protein is CD83, 4-1BB ligand or cytokine.

132. The composition of claim 130, wherein the tumor antigen is mucin 1 (MUC1), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (Her2 / neu), telomerase (TERT), survivin, melanoma-associated antigen A1 (MAGE-A1), MAGE-A3, Wilms tumor 1 (WT1), melanoma preferentially expressed antigen (PRAME), New York esophageal 1 (NY-ESO-1), tumor protein p53, tyrosinase, S100 protein, melanoma-associated antigen recognized by T cells 1 (MART-1), or any combination thereof.

133. The composition of claim 130, wherein the one or more nucleic acids encoding the tumor antigen are chemically modified.

134. The composition of claim 133, wherein the chemical modification comprises a poly A tail, a chemically modified nucleobase, or a 5' end cap.

135. The composition of claim 130, wherein the one or more carriers are complexed with the one or more nucleic acids.

136. The composition of claim 130, wherein the one or more carriers are complexed with the one or more nucleic acids.

137. The composition of claim 130, wherein the one or more nucleic acids are encapsulated by the one or more carriers.

138. The composition of claim 130, wherein the one or more nucleic acids encoding the tumor antigens are encapsulated by the one or more carriers.

139. The composition of claim 130, wherein the composition comprises two or more nucleic acids, wherein the two or more nucleic acids are operably linked to each other.

140. The composition of claim 130, wherein the one or more nucleic acids comprise a sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 2 to 175.

141. The composition of claim 130, wherein the innate immune response induced in the subject is greater than the innate immune response in a subject not administered an effective amount of the composition.

142. A composition comprising: (a) a nucleic acid sequence selected from any one of SEQ ID NOs: 2 to 175; and (b) a delivery vehicle, wherein the delivery vehicle is selected from a diluent, an aqueous solution, a particle, an emulsion, a suspension, a lipid carrier, a vesicle, a yeast cell, or any combination thereof.

143. A composition comprising: a synthetic protein comprising at least one protein construct comprising an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOs: 2 to 175.

144. A composition comprising: (a) a yeast cell, wherein the yeast cell comprises a permeable cell wall; and (b) a nucleic acid encoding a TLR4 agonist, a TLR2 agonist, a TLR5 agonist or a combination thereof.

145. A composition as described in claim 144, wherein the composition further comprises an RNA sequence encoding a viral antigen.

146. The composition of claim 144, wherein the nucleic acid comprises a sequence at least 85% identical to any one of SEQ ID NOs: 2 to 175.

147. The composition of claim 144, wherein the nucleic acid comprises a sequence at least 90% identical to any one of SEQ ID NOs: 2 to 175.

148. The composition of claim 144, wherein the nucleic acid comprises a sequence at least 95% identical to any one of SEQ ID NOs: 2 to 175.

149. The composition of claim 144, wherein the nucleic acid comprises any one of SEQ ID NOs: 2 to 175.

150. The composition of claim 144, wherein the nucleic acid encodes an amino acid sequence that is at least 85% identical to a sequence selected from any one of SEQ ID NOs: 176 to 217.

151. The composition of claim 144, wherein the nucleic acid is encapsulated within, or complexed with, the yeast cell.

152. The composition of claim 144, further comprising enteric-coated microspheres, wherein the yeast cells are encapsulated within the enteric-coated microspheres.

153. The composition of claim 144, wherein the composition is formulated for oral administration to a subject.

154. A vector comprising a nucleic acid encoding: A TLR5 agonist or a functional fragment thereof; a TLR4 agonist or a functional fragment thereof; a TLR2 agonist or a functional fragment thereof; or a combination thereof, wherein the functional fragment is capable of activating the TLR5, TLR4 or TLR2 pathway in a cell when in contact with the cell.

155. The vector of claim 154, wherein the vector comprises a viral vector.

156. The vector of claim 155, wherein the viral vector comprises an adenovirus, an adeno-associated virus (AAV), a recombinant AAV (rAAV), a lentivirus, an alphavirus, a flavivirus, a rhabdovirus, a Kunjin virus, a measles virus, a Lassa virus, or a virus-like particle thereof.

157. The vector of claim 154, wherein the TLR5 agonist comprises a functional derivative of a bacterial movement protein.

158. The vector of claim 154, wherein the TLR5 agonist comprises a DNA sequence encoding an RNA sequence at least 85% identical to any one of SEQ ID NOs: 2 to 37.

159. The vector of claim 154, wherein the TLR5 agonist comprises a sequence at least 85% identical to any one of SEQ ID NOs: 2 to 37.

160. The vector of claim 154, wherein the TLR4 agonist high mobility group protein B1 (HMGB1) protein, nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, transglycosylase, 50S ribosomal protein, heparin-binding hemagglutinin protein, YadA family autotransporting adhesin, dnaJ protein, pneumolysin protein, Omp19 protein, 6,7-dimethyl-8-ribosylcytosine synthase, or any combination thereof.

161. The vector of claim 154, wherein the nucleic acid comprises a DNA sequence encoding an RNA sequence at least 85% identical to any one of SEQ ID NOs: 38 to 103.

162. The vector of claim 154, wherein the nucleic acid comprises a sequence at least 85% identical to any one of SEQ ID NOs: 38 to 103.

163. The vector of claim 154, wherein the TLR2 agonist comprises an outer membrane protein (Omp), a Panton-Valentine two-component leukocidin (PVL) protein, a porin, a TRAP transporter, a hemagglutinin protein, an oxidoreductase protein, or a type VII secretion system.

164. The vector of claim 154, wherein the nucleic acid comprises a DNA sequence encoding an RNA sequence at least 85% identical to any one of SEQ ID NOs: 104 to 175.

165. The vector of claim 154, wherein the nucleic acid comprises a sequence at least 85% identical to any one of SEQ ID NOs: 104 to 175.

166. The vector of claim 154, wherein the vector further comprises a nucleic acid sequence encoding a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

167. The vector of claim 166, wherein the viral antigen comprises a SARS-CoV-2 spike protein.

168. A pharmaceutical composition comprising the composition of any one of claims 1 to 153; and a pharmaceutically acceptable excipient.

169. A pharmaceutical composition comprising the carrier of any one of claims 154 to 167; and a carrier or a pharmaceutically acceptable excipient.

170. A vaccine composition comprising: (a) lipid carrier; (b) one or more RNA adjuvants, wherein the one or more RNA adjuvants comprise a sequence that is at least 85% identical to any one of SEQ ID NOs: 2 to 175; and (c) one or more RNA sequences encoding an antigen.

171. A method of stimulating an immune response in a subject, the method comprising: An effective amount of the composition of any one of claims 1 to 153, the vector of any one of claims 154 to 167, the pharmaceutical composition of claims 168 and 169, or the vaccine composition of claim 170 is administered to the subject to stimulate the immune response.

172. A method of enhancing an immune response to an antigen encoded by RNA in an RNA vaccine composition, the method comprising: (a) administering to a subject an effective amount of the composition of any one of claims 1 to 153, the vector of any one of claims 154 to 167, the pharmaceutical composition of claims 168 and 169, or the vaccine composition of claim 170; and (b) administering an RNA vaccine composition to the subject, wherein the RNA vaccine composition comprises RNA encoding an antigen, thereby enhancing the immune response to the antigen encoded by the RNA.

173. A method of treating an infection in a subject, the method comprising: Administering an effective amount of the composition of any one of claims 1 to 153 to a subject, thereby treating the infection in the subject.

174. The method of any one of claims 171 to 173, wherein the administration is systemic administration or local administration.

175. The method of any one of claims 171 to 174, wherein the administration is intramuscular, intravenous, oral, intradermal, intratumoral, inhaled, subcutaneous, or intraperitoneal.

176. The method of any one of claims 171 to 175, wherein the administration is at least about once every 24 hours, every 168 hours (7 days), every 720 hours (30 days), or every 8760 hours (365 days, 1 year).

177. The method of any one of claim 171 or claim 173, wherein the method further comprises administering an RNA vaccine composition to the subject.

178. The method of claim 171 or claim 172, wherein the subject has an infection or is at risk of developing an infection.

179. The method of claim 173 or claim 178, wherein the infection is a viral infection.

180. The method of claim 179, wherein the viral infection is an upper respiratory tract viral infection.

181. The method of claim 180, wherein the upper respiratory tract viral infection is COVID-19, SARS infection, MERS infection, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia, or rhinovirus infection.

182. The method of claim 173 or claim 178, wherein the infection is a bacterial infection, a fungal infection, a parasitic infection, or a yeast infection.

183. The method of claim 172, wherein the antigen is a spike protein, a glycoprotein, a viral envelope protein, or a tumor antigen.

184. The method of claim 183, wherein the spike protein is from the SARS-CoV-2 virus.

185. The method of any one of claims 171 to 184, wherein the subject is a mammal.

186. The method of any one of claims 171 to 185, wherein the subject is a human.

187. The method of any one of claims 171-186, wherein an additional dose of the composition or the RNA vaccine composition is administered to the subject.

188. A method of treating cancer in a subject, the method comprising: Administering an effective amount of the composition of any one of claims 1 to 153 to a subject, thereby treating cancer in the subject.

189. The method of claim 188, wherein the administration is systemic administration or local administration.

190. The method of claim 188 or claim 189, wherein the administration is intramuscular, intravenous, oral, intranodal, intrasplenic, intradermal, intratumoral, inhaled, subcutaneous, or intraperitoneal.

191. The method of any one of claims 188 to 190, wherein the administration is at least about once every 24 hours, every 168 hours (7 days), every 720 hours (30 days), or every 8760 hours (365 days, 1 year).

192. The method of any one of claims 188 to 191, wherein the method further comprises administering an RNA vaccine composition to the subject.

193. The method of any one of claims 188 to 192, wherein the subject has cancer, has been diagnosed with cancer, or is at risk of developing cancer.

194. The method of claims 188-193, wherein the subject has a solid tumor or a blood cancer.

195. The method of claim 193, wherein the cancer is lung cancer, breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, lung cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, stomach cancer, skin cancer, head and neck cancer, or thyroid cancer.

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