RNA Adjuvant, Method and Use

Abstract

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

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 347,635, filed on June 1, 2022, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing XML filed via the USPTO Patent Electronic Filing System. This Sequence Listing XML, entitled 207481 - 701601_SL.xml, was created on May 31, 2023 and is 427,686 bytes in size.

Background Art

[0003] RNA vaccines have emerged as promising therapeutics for preventing the spread of infectious diseases such as COVID - 19. However, RNA vaccines are costly, require high doses of RNA per vaccine, and lack long - term effectiveness for preventing infectious diseases. Furthermore, RNA is translated using host cell machinery regulated by the interferon system, which can reduce the amount of RNA translated in vivo. Additionally, current vaccine compositions can cause unwanted side effects. Therefore, there is a need for an adjuvant that can reduce the side effects of RNA vaccines, optimize the RNA dosage in RNA vaccines, and promote the innate and adaptive immune responses that enhance the effectiveness of RNA vaccines.

Summary of the Invention

[0004] Provided herein is a composition for inducing an immune response following administration to a subject, the composition comprising: (a) a nucleic acid (e.g., RNA, DNA, or an RNA adjuvant) encoding a synthetic protein, the synthetic protein comprising at least a functional fragment of a toll-like receptor 5 (TLR5) agonist capable of activating the TLR5 pathway in a cell upon contact with the cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein administration of an effective amount of the composition to the subject in the presence of an antigen induces a first innate immune response in the subject.

[0005] Provided herein is a composition comprising: (a) a carrier (e.g., a lipid carrier); and (b) an RNA sequence encoding at least a functional fragment of a bacterial motility protein, the functional fragment of the bacterial motility protein being a TLR5 agonist, wherein in some embodiments, the functional fragment activates the TLR5 pathway in a cell upon contact with the cell.

[0006] Provided herein is a composition for inducing an immune response following administration to a subject, the composition comprising: (a) a nucleic acid (e.g., RNA, DNA, or an RNA adjuvant) encoding a synthetic protein, the synthetic protein comprising at least a functional fragment of a toll-like receptor 4 (TLR4) agonist capable of activating the TLR4 pathway in a cell upon contact with the cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein administration of an effective amount of the composition to the subject in the presence of an antigen induces a first innate immune response in the subject.

[0007] As used herein, there is provided a composition comprising: (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, and the functional fragment of the TLR4 agonist is capable of activating the TLR4 pathway in a cell upon contact with the cell.

[0008] As used herein, there is provided a composition for inducing an immune response following administration to a subject, the composition comprising: (a) a nucleic acid (e.g., RNA, DNA, or an RNA adjuvant) encoding a synthetic protein, the synthetic protein comprising at least a functional fragment of a toll-like receptor 2 (TLR2) agonist capable of activating the TLR2 pathway in a cell upon contact with the cell; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein administration of an effective amount of the composition to the subject in the presence of an antigen induces a first innate immune response in the subject.

[0009] As used herein, there is provided a composition comprising: (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, and the functional fragment is capable of activating the TLR2 pathway in a cell upon contact with the cell.

[0010] Provided herein is a composition for inducing an immune response upon administration to a subject, the composition comprising: (a) one or more carriers (e.g., lipid carriers); (b) one or more nucleic acids (e.g., RNA or DNA) encoding (i) at least a 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. Following administration of an effective amount of the composition, a innate immune response is induced in the subject in the presence of the viral antigen, thereby inducing an immune response against the viral antigen. In some embodiments, the delivery vehicle is formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration.

[0011] Provided herein is a composition 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 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.

[0012] Provided herein is a composition 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 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.

[0013] As used herein, a composition is provided that comprises (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 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. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 80% identical to SEQ ID NO: 206. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 80% identical to SEQ ID NO: 210. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 212. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 85% identical to SEQ ID NO: 213. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 215. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 217.

[0014] As used herein, a composition is provided that comprises (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 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] As used herein, a composition is provided that comprises a synthetic protein comprising at least one protein construct, wherein the at least one protein construct comprises an amino acid sequence selected from SEQ ID NOs: 176 to 217. Further provided herein are compositions comprising two or more, three or more, four or more, or five or more protein constructs.

[0016] As used herein, there is provided a composition comprising a nucleic acid encoding a synthetic protein comprising at least one protein construct, wherein the at least one protein construct comprises an amino acid sequence selected from SEQ ID NOs: 176-217. Further provided herein are compositions comprising two or more, three or more, four or more, or five or more protein constructs.

[0017] As used herein, there is provided a composition comprising (a) a yeast cell comprising a permeable cell wall, and (b) one or more nucleic acids (e.g., DNA, RNA, or a combination thereof) encoding at least a functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof, wherein the functional fragment activates the TLR4, TLR5, or TLR2 pathway in the cell upon contact with the cell.

[0018] As used herein, there is provided a composition for inducing an immune response upon administration to a subject, the composition comprising (a) one or more carriers (e.g., lipid carriers), (b) one or more nucleic acids encoding (i) at least a 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 administration of an effective amount of the composition induces an innate immune response in the subject in the presence of the tumor antigen, thereby inducing an immune response against the tumor antigen. In some embodiments, the functional fragment activates the TLR4, TLR5, or TLR2 pathway in the cell upon contact with the cell.

[0019] As used herein, there is provided a vector comprising one or more nucleic acids (e.g., DNA, RNA, or a combination thereof) encoding a TLR5 agonist, a TLR4 agonist, a TLR2 agonist, or a combination thereof.

[0020] In this specification, a composition is provided, which comprises a vector provided herein and a carrier (e.g., a lipid carrier).

[0021] In this specification, a vaccine composition is provided, which comprises the composition provided herein and a pharmaceutically acceptable excipient.

[0022] In this specification, a vaccine composition is provided, which comprises (a) 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 the sequences of SEQ ID NOs: 2-175, and (c) one or more RNA sequences encoding an antigen. Further provided herein is a vaccine composition, wherein each of the one or more RNA sequences encoding an antigen encodes a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or any combination thereof.

[0023] In this specification, a vaccine composition is provided, which comprises (a) a lipid carrier, (b) one or more nucleic acids encoding the RNA adjuvant provided herein, wherein the one or more RNA adjuvants comprise a sequence that is at least 85% identical to any one of the sequences of SEQ ID NOs: 2-175, and (c) one or more nucleic acids encoding an antigen. Further provided herein is a vaccine composition, wherein each of the one or more nucleic acids encoding an antigen encodes a viral antigen, a bacterial antigen, a fungal antigen, a parasitic 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 the RNA adjuvant are DNA, RNA, or a combination thereof.

[0024] This specification provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of a composition provided herein, a vector provided herein, or a vaccine composition provided herein, whereby the immune response is stimulated. This specification provides a method for enhancing an immune response against an antigen encoded by a nucleic acid (e.g., RNA) in a vaccine composition (e.g., an RNA vaccine), the method comprising: (a) administering to the 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 comprising RNA encoding the antigen, whereby the immune response against the antigen encoded by the RNA is enhanced. This specification provides a method for treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of a composition provided herein, a vector provided herein, or a vaccine composition provided herein, whereby the infectious disease in the subject is treated.

[0025] The novel features of the invention are particularly pointed out in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description, which describes exemplary embodiments in which the principles of the invention are utilized, and from the accompanying drawings.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0033] Hereinafter, various aspects will be described more fully. However, such aspects may be embodied in many different forms and should not be construed as limited to the embodiments described herein. On the contrary, these embodiments are provided so that this disclosure will be complete and will fully convey the scope to those skilled in the art.

[0034] This specification provides RNA adjuvant compositions and vaccine compositions for inducing an immune response against an antigen, methods thereof, and uses thereof for the prevention of infectious diseases and cancer treatment. Briefly stated, this specification further describes (1) RNA adjuvant compositions, (2) nucleic acids encoding an antigen, (3) lipid carriers, (4) yeast cell compositions, (5) pharmaceutical vaccine compositions, dosage, and administration, and (6) therapeutic uses.

[0035] Definitions All definitions defined and used in this specification shall be understood to take precedence over the definitions in the dictionary of the defined terms, the definitions in the documents incorporated by reference, and / or their ordinary meanings.

[0036] All references, patents, and patent applications disclosed in this specification are incorporated by reference with respect to the subject matter of the invention to which each is cited, which may in some cases include the entire document. All references, including patent references and non-patent references, disclosed in this specification are incorporated by reference in their entirety into this specification as if each were individually incorporated. However, it should be understood that when a patent, patent application, or publication containing an explicit definition is incorporated by reference, such explicit definition applies to the incorporated patent, patent application, or publication in which it is found and not necessarily to the text of this application, particularly the claims of this application. In such cases, the definitions provided in this specification are intended to supersede.

[0037] As used herein in this specification and in the claims, the indefinite articles "a" and "an" shall be understood to mean "at least one" unless a clear indication to the contrary is given.

[0038] As used herein and in the claims, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined (i.e., elements that coexist in some instances and exist separately in other instances). Multiple elements listed with "and / or" should be construed in the same manner (i.e., "one or more" of the elements so conjoined). Other elements may optionally be present, whether or not they are related to the specifically identified elements, in addition to the elements specifically identified by the "and / or" clause. Thus, by way of non-limiting example, reference to "A and / or B" when used in conjunction with an open-ended phrase such as "comprising" may, in one embodiment, refer to only A (optionally including elements other than B), in another embodiment, refer to only B (optionally including elements other than A), and still in another embodiment, refer to both A and B (optionally including other elements), and so on.

[0039] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be construed as inclusive (i.e., including at least one of several elements or a list of elements, as well as two or more, and optionally, additional unlisted items). Only terms such as "only one of...", "exactly one of...", or when used in the claims, "consisting of...", which have a clear indication to the contrary, will refer to the inclusion of exactly one element from several elements or a list of elements. Generally, the term "or" as used herein shall be construed to indicate an exclusive alternative (i.e., "either one or the other, but not both") only when preceded by an exclusive term such as "any one of...", "one of...", "only one of...", or "exactly one of...". "Consisting essentially of...", when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0040] As used herein, the term "optional" or "optionally" means that the situation described thereafter may or may not occur, whereby the description includes cases where the situation occurs and cases where it does not occur.

[0041] As used herein, the term "about" or "approximately" means within a range of ±20% of a given value. Alternatively, especially with respect to biological systems or processes, this term may mean within one order of magnitude, preferably within a factor of two, of the value. When a specific value is recited herein and in the claims, unless otherwise specified, the term "about" is implicit and means within the error range acceptable for that specific value in this context.

[0042] The terms "effective amount" or "therapeutically effective amount" refer to an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0043] (1) RNA adjuvant composition Provided herein are compositions comprising nucleic acids. Further provided herein are compositions comprising an RNA adjuvant. Further provided herein are compositions (e.g., pharmaceutical compositions and vaccine compositions), wherein the vaccine composition comprises a nucleic acid (e.g., an RNA adjuvant provided herein) and a nucleic acid encoding an antigen (e.g., RNA). In some embodiments, the nucleic acids provided herein encode proteins that activate toll-like receptors (TLRs). In some embodiments, the nucleic acid activates a TLR. TLRs are broadly classified into two subfamilies, cell surface TLRs and intracellular TLRs, based on their localization. Cell surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, while intracellular TLRs are localized to endosomes and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13. Cell surface TLRs generally recognize components of microbial membranes such as lipids, lipoproteins, and proteins. For example, TLR4 recognizes bacterial lipopolysaccharide (LPS). TLR2, together with TLR1 or TLR6, recognizes a variety of PAMPs including lipoproteins, peptidoglycan, lipoteichoic acid, zymosan, mannan, and tGPI-mucin. TLR5 recognizes the bacterial flagellin protein. Human TLR10, in conjunction with TLR2, recognizes ligands from Listeria. TLR10 can also sense influenza A virus infection.

[0044] TLRs recognize pathogen-associated molecular patterns (PAMPs) derived from microorganisms and signal through the recruitment of specific adapter molecules, leading to the activation of transcription factors (e.g., NF-κB), thereby regulating the innate immune response against the PAMP or the microorganism expressing the PAMP. The innate immune response is the first line of defense against invading pathogens. The innate immune response is an antigen-independent 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 thus cannot recognize or "remember" the body if it is ever exposed to the same pathogen in the future. Adaptive immunity, on the other hand, is antigen-dependent and antigen-specific and thus involves a time lag between antigen exposure and maximal response. A prominent feature of adaptive immunity is the ability to remember, which enables the host to mount a more rapid and efficient immune response when subsequently exposed to the antigen. Innate and adaptive immunity are not mutually exclusive host defense mechanisms but are complementary, and deficiencies in either system result in host vulnerability or inappropriate responses.

[0045] The advantage of the compositions provided herein is that the RNA adjuvant enhances the innate immune signaling pathway via TLR signaling while promoting the adaptive immune response. This effect reduces the effective amount of RNA required to encode an antigen in the vaccine composition, and thus can reduce the dosage required for the mRNA encoding the antigen as compared to a vaccine composition without an mRNA adjuvant. The mRNA adjuvants provided herein activate the signaling of nuclear factor-kappa B (NF-κB) downstream of TLRs, which is required to stimulate T cell activation. The TLR5-mediated activation of NF-κB, unlike other TLRs, is mainly limited to epithelial and endothelial cells and is due to the unique tissue specificity of TLR5 expression without T cells and macrophages, resulting in a relatively low degree of production of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β). Since TNF-α and IL-1β can cause negative side effects such as septic shock, fever, and irritation at the administration site, this contributes to the safety of mRNA vaccines in combination with adjuvants based on TLR5 agonists. Furthermore, the mRNA adjuvants provided herein induce and promote an antigen-targeted adaptive immune response and long-term memory of pathogens by the immune system.

[0046] The nucleic acids provided herein (e.g., RNA adjuvants) encode TLR agonists or functional fragments thereof. The functional fragments can activate the TLR pathway or TLRs in cells upon contact with the cells. In some embodiments, the TLR agonist binds directly to the TLR. In some embodiments, the TLR agonist or its functional fragment is a naturally occurring TLR agonist, protein, or nucleic acid. In some embodiments, the TLR agonist or its functional fragment is a non-naturally occurring TLR agonist, protein, or nucleic acid. In some embodiments, the non-naturally occurring TLR agonist is an RNA sequence encoding a TLR agonist protein. In some embodiments, the TLR agonist or its functional fragment is a synthetic protein (also known as an artificial protein). The synthetic proteins provided herein do not occur naturally and can be engineered using known methods, such as molecular cloning techniques. In some embodiments, the compositions provided herein comprise a plurality of the mRNA adjuvants provided herein. In some embodiments, the mRNA adjuvants provided herein activate TLR2, TLR4, and / or TLR5.

[0047] In some embodiments, the activation of TLRs provided herein increases the level or activity of NF-κB. NF-κB is a transcription factor that regulates the cellular response to infectious agents and acts as a mediator of innate and adaptive immune responses. NF-κB also mediates the production of inflammatory cytokines.

[0048] In some embodiments, the compositions provided herein include at least a functional fragment of a TLR5 agonist, which TLR agonist includes a functional fragment of a bacterial motility protein. In some embodiments, the bacterial motility protein is a bacterial flagellin protein, its derivative, or a functional fragment thereof. In some embodiments, the bacterial motility proteins provided herein are a truncated form of a bacterial flagellin protein, a secreted form of a bacterial flagellin protein, or a combination thereof. Flagellin contains two to four structural domains. The common D0 and D1 domains are embedded in the core of the flagellar filament by mediating flagellin-flagellin interactions and are conserved among bacterial species due to their functional importance in filament formation. The flagellin D1 domain has a common molecular pattern for interaction with TLR5 despite sequence and domain differences among flagellins. The D0 and D1 domains stimulate TLR5 activation. In three-domain and four-domain flagellins, the D1 domain extends to auxiliary domains (D2 and D3) located on the surface of the flagellar filament. In contrast to D0 and D1, the D2 or D3 domains exhibit considerable sequence and structural diversity and are thought to activate adaptive immunity. Many Gram-positive bacteria, such as Bacillus subtilis and Clostridium difficile, etc., lack hypervariable domains and thus express flagellin that contains the minimal regions (D0 and D1 domains) necessary for TLR5 activation and flagellin polymerization into flagellar filaments. Surprisingly, not all bacterial flagellins activate TLR5. For example, flagellins from Campylobacter jejuni and Helicobacter pylori, which belong to ε-proteobacteria, do not induce TLR5 signaling and can escape the TLR5-mediated immune surveillance mechanism. The major functional difference between TLR5-activating flagellins and non-activators can be attributed to sequence diversity.

[0049] The proteins provided herein (e.g., synthetic proteins or proteins encoded by the nucleic acids provided herein) can include two (D0 and D1), three (D0, D1, and D2), or four (D0, D1, D2, and D3) domains derived from bacterial flagellin. Compositions are provided herein in which a functional fragment of a toll-like receptor 5 (TLR5) agonist comprises the D0 region and the D1 region of a bacterial flagellin protein. In some embodiments, a functional fragment of a toll-like receptor 5 (TLR5) agonist comprises the D0 region, the D1 region, and the D2 region of a bacterial flagellin protein. In some embodiments, a 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 a bacterial flagellin protein.

[0050] In some embodiments, the compositions provided herein comprise at least a functional fragment of a TLR4 agonist. In some embodiments, the TLR4 agonist comprises at least a functional fragment of a high mobility group box 1 (HMGB1) protein, nicotinic acid nucleotide--dimethylbenzimidazole phosphoribosyltransferase, transglycosylase, 50S ribosomal protein, heparin-binding hemagglutinin protein, YadA family autotransporter adhesin, dnaJ protein, pneumolysin protein, Omp19 protein, 6,7-dimethyl-8-ribityllumazine synthase, or any combination thereof. In some embodiments, the functional fragment activates the TLR4 pathway in cells upon contact with the cells.

[0051] In some embodiments, the compositions provided herein comprise at least a functional fragment of a TLR2 agonist. In some embodiments, the TLR2 agonist comprises an outer membrane protein (Omp), a Panton-Valentine leukocidin (PVL) protein, a porin protein, a TRAP transporter protein, a hemagglutinin protein, an oxidoreductase protein, or at least a functional fragment of a type VII secretion system. In some embodiments, the functional fragment activates the TLR2 pathway in cells upon contact with the cells.

[0052] The proteins encoded by the nucleic acids provided herein (e.g., DNA sequences, RNA sequences, or RNA adjuvants) can be designed from the amino acid sequences of any bacterial species or mammal that encodes an immune-stimulatory pathway. The synthetic proteins provided herein can be chimeric proteins that comprise fragments of proteins from two or more different organisms to form a functional TLR agonist that activates a TLR receptor, such as human TLR5, human TLR4, or human TLR2.

[0053] The proteins encoded by the RNA adjuvants or nucleic acids provided herein may include modifications that improve protein stability, targeting, or function. In some embodiments, the protein includes a signal peptide for targeted intracellular localization. In some embodiments, the proteins provided herein further include the signal sequence of MRSLSVLALLLLLLLAPASA (SEQ ID NO: 1). In some embodiments, the proteins provided herein do not include a signal sequence. In some embodiments, the proteins provided herein further include an N-terminal spoke region. In some embodiments, the N-terminal spoke region includes the sequence of SGLRINSAKDDA (SEQ ID NO: 218). In some embodiments, the proteins provided herein further include a linker. A linker 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 linker can be configured according to specific requirements, such as stability or length between the first protein construct and the second protein construct. As another example, the linker can be configured to have sufficient length and flexibility. In some embodiments, the linker can be configured to facilitate the expression and purification of the proteins provided herein. In some embodiments, the linker includes the sequence of SPG. In some embodiments, the protein includes a C-terminal spoke region. In some embodiments, the C-terminal spoke region includes the sequence of EDADYA (SEQ ID NO: 220). In some embodiments, the proteins provided herein include a thrombin cleavage site. In some embodiments, the thrombin cleavage site includes the sequence of LVPRGS (SEQ ID NO: 221). In some embodiments, the proteins provided herein include a peptide tag. In some embodiments, the peptide tag is a sequence including a histidine (His) tag (e.g., HHHHHH (SEQ ID NO: 222)).

[0054] The compositions provided herein may include any one of the RNA adjuvants or sequences listed in Table 1, derivatives, variants, or functional fragments thereof. The compositions provided herein may also include DNA sequences encoding the RNA adjuvants or amino acid sequences listed in Table 1, derivatives, variants, or functional fragments thereof. In some embodiments, the RNA adjuvants provided herein encode synthetic proteins and / or TLR agonists, derivatives, or functional fragments thereof that include at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the sequences listed in Table 1. In some embodiments, the nucleic acid sequences or amino acid sequences provided herein include sequences that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similar to the sequences listed in Table 1. In some embodiments, the RNA sequences provided herein include the same sequence as the sequences listed in Table 1 or encode amino acid sequences that are identical to the sequences listed in Table 1. The percent sequence identity (or similarity) relative to a reference sequence for a given sequence is defined as the percentage of identical residues determined after aligning the two sequences and introducing gaps if necessary to obtain the maximum percent sequence identity. The percent identity can be calculated using alignment methods known in the art, for example, sequence alignment can be performed using publicly available software such as BLAST, Align, ClustalW2, etc. One of ordinary skill in the art can determine appropriate parameters for the alignment, but the default parameters for BLAST are specifically contemplated.

[0055] 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 modification(s) can include, for example, substitution, deletion, insertion, chemical modification of one or more nucleobases, or chemical modification to the phosphate backbone, nucleotide, nucleobase, or nucleoside. Such modifications can be made to the sequences of the RNA adjuvants provided herein, the DNA sequences encoding the sequences of the RNA adjuvants, or any sequence disclosed herein (e.g., the mRNA encoding an antigen). Methods for modifying nucleic acid or amino acid sequences are known. One of ordinary skill in the art will understand that the modification(s) can be located at any position(s) of the nucleic acid such that the function of the nucleic acid or synthetic protein is not substantially reduced. For example, software can be used to match identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. The nucleic acids provided herein can be prepared according to any available technique including, but not limited to, chemical synthesis, enzymatic synthesis (which is collectively referred to as in vitro transcription), cloning, enzymatic cleavage, or chemical cleavage. In some cases, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can be present at various positions within the nucleic acid.

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[0056] In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence that is at least 90% identical to any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence that is at least 95% identical to any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise a sequence selected from any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise SEQ ID NO: 2, its variants, or functional fragments. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise SEQ ID NO: 62, its variants, or functional fragments. In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) comprise SEQ ID NO: 98, its variants, or functional fragments.

[0057] In some embodiments, the nucleic acids provided herein (e.g., RNA adjuvants) encode an amino acid sequence that is at least 75% identical 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 NO: 176, SEQ ID NO: 181, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, its variants, or functional fragments. In some embodiments, the compositions provided herein include a protein comprising an amino acid sequence that is 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% identical to any one of SEQ ID NOs: 176 to 204. In some embodiments, the compositions provided herein include a protein comprising an amino acid sequence that is 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 include a nucleic acid encoding a protein comprising an amino acid sequence that is 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.

[0058] Also provided herein are compositions 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 linker between each protein construct. In some embodiments, the composition comprises one or more protein constructs selected from Table 2. [Table 2]

[0059] In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 75% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 80% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 85% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 90% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 95% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 96% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 97% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that comprises at least 98% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217.In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence that contains at least 99% sequence identity to a sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode an amino acid sequence selected from any one of SEQ ID NOs: 205-217. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) encode a leader peptide sequence. In some embodiments, the leader peptide sequence contains the amino acid sequence of MRSLSVLALLLLLLLAPASA (SEQ ID NO: 223). In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or combinations thereof) further encode a zipper. For example, the zipper for a trimeric peptide may contain the amino acid sequence of RMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO: 224), and the zipper for a dimer may contain the amino acid sequence of RMKQLEDKIEELLSKIYHLENEIARLKKLIGER (SEQ ID NO: 225).

[0060] This specification provides a synthetic protein comprising at least one protein construct or a nucleic acid encoding at least one protein construct, wherein the at least one protein construct comprises an amino acid sequence that is 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% identical to a sequence selected from SEQ ID NOs: 176 - 217. In some embodiments, the synthetic protein provided herein comprises a sequence that is 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% identical to SEQ ID NO: 206, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, or a variant thereof. This specification further provides a composition 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 DNA encoding a self - replicating RNA sequence.

[0061] (2) Nucleic acid encoding an antigen This specification provides a composition comprising one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise deoxyribonucleic acid (DNA). In some embodiments, the one or more nucleic acids comprise ribonucleic acid (RNA). In some embodiments, the one or more nucleic acids comprise a mixture of DNA and RNA.

[0062] In some embodiments, the nucleic acids provided herein comprise the sequences of the RNA adjuvants provided herein (e.g., encoding TLR agonist proteins) and an antigen. In some embodiments, the nucleic acids provided herein comprise polynucleotides that include one or more modified nucleotides or nucleobases, and / or analogs thereof. The polynucleotides provided herein can include modified nucleotides such as methylated nucleotides and their analogs. When present, modifications to the nucleotide structure can be imparted before or after the assembly of the compositions provided herein. Modified nucleosides and nucleotides that can be incorporated and modified nucleobases that can be present in the RNA molecule include 1-methyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 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, under modified hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, 4-demethylwyosine, epoxyqueuosine, hydroxywybutosine, 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-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 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, methyl ester of uridine 5-oxyacetic acid, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-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 are included. In some embodiments, the nucleic acids provided herein include pseudouridine modifications to ensure the stability of mRNA. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the nucleic acid can contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. RNA sequences or DNA sequences encoding RNA adjuvants can be modified with respect to their codon usage frequency, for example, to increase the translational efficiency and half-life of the RNA. To increase the half-life of the RNA, a polyA tail (e.g., of about 30 adenosine residues or more) can be attached to the 3' end of the RNA. A cap structure can provide stability and translational efficiency to the RNA molecule. In some embodiments, the RNA adjuvants provided herein or the DNA encoding the RNA adjuvants are chemically modified. In some embodiments, the nucleic acid encoding the antigen is chemically modified. In some embodiments, the nucleic acid encoding the antigen contains DNA. In some embodiments, the nucleic acid encoding the antigen contains RNA. In some embodiments, the chemical modifications to the RNA sequences provided herein include a poly-A tail, the chemically modified nucleobases provided herein, or a 5' end cap.,

[0063] In some embodiments, the RNA provided herein is mRNA. In some embodiments, the mRNA is non-replicating mRNA. In some embodiments, the mRNA is self-amplifying mRNA. Non-replicating mRNA and self-amplifying mRNA can utilize the host cell's translation machinery for the production of antigen targets and the initiation of an adaptive immune response. Non-replicating mRNA encodes the protein antigen(s) of interest, while self-amplifying mRNA can also encode proteins that enable RNA replication.

[0064] As used herein, there is provided a composition comprising RNA encoding an antigen, or a plurality of RNAs each encoding a different antigen. The compositions provided herein can be multivalent for use as a vaccine to induce an immune response against a plurality of antigens (e.g., viral, bacterial, and / or tumor antigens). Further provided herein is a composition comprising an RNA adjuvant provided herein and RNA encoding an antigen. In some embodiments, the RNA sequence encoding the antigen is operably linked to the RNA adjuvant. Further provided herein is a composition comprising an RNA adjuvant provided herein and a protein antigen or fragment thereof. The protein antigen can include an inactivated bacterium or virus, a fragment of a bacterial toxin, or a fragment of an antigen expressed by a microorganism.

[0065] In some embodiments, the antigen provided herein is derived from a microorganism. In some embodiments, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen.

[0066] In some embodiments, the antigens provided herein are bacterial antigens. Non-limiting examples of infectious bacteria include E. coli, Pseudomonas aeruginosa, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, 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 species), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Brucella abortus, Pasteur ella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Nocardia brasiliensis, Borrelia hermsii, Borrelia burgdorferi, and Actinomyces israelii.

[0067] In some embodiments, the antigen provided herein is a viral antigen. In some embodiments, the antigen comprises a spike protein, a glycoprotein, a hemagglutinin protein, or a viral envelope protein. In some embodiments, the viral antigen is derived from an adenovirus, an astrovirus, a bocavirus, a bunyavirus, 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. In some embodiments, the SARS virus is the SARS-CoV-2 virus.Further non-limiting examples of viruses include Retroviridae (e.g., HIV); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus, West Nile virus, Zika 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., hantavirus, bunyavirus, phlebovirus, and nairovirus); Arenaviridae (hemorrhagic fever virus); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (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 (e.g., African swine fever virus); cytomegalovirus (CMV) pneumonia, enteritis, and retinitis; Epstein-Barr virus (EBV) lymphoproliferative disorders; varicella-zoster virus (VZV); HSV-1 and -2 mucositis; HSV-6 encephalitis, BK virus hemorrhagic cystitis; and hepatitis A, B, or C.

[0068] Exemplary viral antigens that can be encoded by the RNA molecules provided herein include spike (S) protein, nucleocapsid protein (NP), hemagglutinin (HA), neuraminidase (NA), Den1 virus envelope protein, Den2 virus envelope protein, Den3 virus envelope protein, Den4 virus envelope, lipopeptide, gene products of the HIV gag, pol, and env genes, Nef protein, reverse transcriptase, nucleoprotein, matrix protein (M1), membrane protein (M2), components of the transcriptase (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 El and E2), glycoprotein B, HPV antigens (e.g., HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35), Epstein-Barr nuclear antigen (EBNA)-l, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), latent membrane protein 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), but are not limited thereto.

[0069] In some embodiments, the antigen provided herein is a fungal antigen. 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, the compositions provided herein include antigens from other infectious organisms. Additional examples of infectious organisms include, but are not limited to, parasitic protozoa such as Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Trichinella spiralis, Strongyloides ratti, and Toxoplasma gondii.

[0070] Also provided herein are compositions for use in the treatment of cancer. In some embodiments, the composition comprises RNA encoding a tumor viral antigen. In some embodiments, the tumor viral antigen is derived from a human papillomavirus (HPV) antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV) antigen, a Merkel cell polyomavirus (MCV) antigen, a human T-cell lymphotropic virus type 1 (HTLV-1) antigen, or an Epstein-Barr virus (EBV) antigen.

[0071] In some embodiments, the compositions provided herein comprise RNA encoding a tumor antigen. In some embodiments, the tumor antigen is a surface protein, a cytosolic protein, or a transmembrane protein. The tumor antigen is a protein expressed by cancer cells. Some tumor antigens, called neoantigens, are proteins that occur in cancer cells in the presence of point mutations, frameshift mutations, and gene rearrangements. The RNA molecules provided herein can encode one 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), preferentially expressed antigen in melanoma (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.

[0072] Furthermore, the RNA molecules provided herein can encode a protein that modulates an immune response against cancer cells or tumors. In some embodiments, the compositions provided herein comprise RNA encoding an immunomodulatory protein. In some embodiments, the composition comprises one or more RNA sequences encoding an immunomodulatory protein. In some embodiments, the immunomodulatory protein is CD83, 4-1BB ligand, a cytokine, or any combination thereof. Exemplary cytokines useful in the treatment of 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.

[0073] The compositions provided herein may further comprise a delivery vehicle. Non-limiting examples of delivery vehicles that may be used include solvents, diluents, aqueous solutions, saline solutions, particles, emulsifiers, surfactants, lipid carriers, lipidoids, artificial vesicles, cells, extracellular vesicles, yeast cells, antibodies, aptamers, vectors, viral vectors, adenoviral vectors, lentiviral vectors, micelles, liposomes, and transfection agents (e.g., polyvalent cationic lipid compositions such as polyethyleneimine (PEI), LIPOFECTIN®, LIPOFECTACE®, LIPOFECTAMINE™, CELLFECTIN®, DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, as well as dendrimer compositions, particularly dendrimers known as dense starburst dendrimers, PAMAM dendrimers, grafted dendrimers, and dendrigrafts, and G5-G10 dendrimers including SUPERFECT®).

[0074] Vectors are provided herein, the vectors comprising one or more nucleic acids encoding a TLR5 agonist provided herein, a TLR4 agonist provided herein, a TLR2 agonist provided herein, or combinations 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 an adenovirus, an adeno-associated virus (AAV), a recombinant AAV (rAAV), a lentivirus, an alphavirus, a flavivirus, a rhabdovirus, a herpes simplex virus, a Kunjin virus, a measles virus, a Lassa virus, or virus-like particles thereof. In some embodiments, the 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., an alphavirus) or a portion thereof.

[0075] 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 vector comprises a DNA sequence encoding an RNA sequence that is at least 85% identical to any one of SEQ ID NOs: 2-37. In some embodiments, the vector comprises a sequence that is at least 85% identical to SEQ ID NO: 226. In some embodiments, the vector comprises a nucleic acid sequence that is at least 85% identical to any one of SEQ ID NOs: 2-37. In some embodiments, the vector comprises a DNA sequence encoding an RNA sequence that is at least 85% identical to any one of SEQ ID NOs: 38-103. In some embodiments, the vector comprises a nucleic acid sequence that is at least 85% identical to any one of SEQ ID NOs: 38-103. In some embodiments, the vector comprises a DNA sequence encoding an RNA sequence that is at least 85% identical to any one of SEQ ID NOs: 104-175. In some embodiments, the vector comprises a nucleic acid sequence that is at least 85% identical to any one of SEQ ID NOs: 104-175. 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 the SARS-CoV-2 spike protein or any antigen provided herein.

[0076] Liposomes and / or nanoparticles can also be used in the administration of the compositions herein. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLV)). MLVs generally have a diameter in the range of 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUV) having a diameter in the range of 200-500 angstroms and containing an aqueous solution in the core.

[0077] Nanoparticle carriers may also be used as delivery vehicles for the RNA molecules provided herein. In some embodiments, the 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 are further considered below.

[0078] (3) Carrier Compositions comprising a carrier are provided herein. The carrier facilitates delivery of the nucleic acids provided herein into cells or tissues. In some embodiments, the carrier is a lipid carrier or a lipid nanoparticle. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or the RNA adjuvants provided herein) are complexed with a lipid carrier. In some embodiments, the nucleic acids provided herein (e.g., DNA, RNA, or the RNA adjuvants provided herein) are encapsulated within a lipid carrier. In some embodiments, the nucleic acids encoding the antigens provided herein are complexed with a lipid carrier. In some embodiments, the nucleic acids encoding the antigens provided herein are encapsulated within a lipid carrier. In some embodiments, the vectors provided herein are complexed with a lipid carrier. In some embodiments, the vectors provided herein are encapsulated within a lipid carrier. In some embodiments, the protein antigens provided herein are complexed with a lipid carrier. In some embodiments, the protein antigens provided herein are encapsulated within a lipid carrier. In some embodiments, the nucleic acids encoding an antigen are encapsulated within the same lipid carrier as an RNA adjuvant, a TLR agonist, or a nucleic acid encoding a TLR agonist. In some embodiments, the RNA encoding an antigen is encapsulated within a lipid carrier different from the RNA adjuvant. Exemplary configurations of carrier-RNA complexes are provided in FIGS. 2A-2D.

[0079] 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 carriers are cationic lipid nanoparticles, ionizable lipid nanoparticles, PEG lipid carriers, polymeric lipid nanoparticles, polymer-conjugated lipid carriers, synthetic vesicles, liposomes, exosomes, endosomes, lipoplexes, lipidoids, derivatives thereof, variants, or any combination thereof.

[0080] In some embodiments, the lipid carrier comprises a cationic lipid. In some embodiments, the lipid carriers provided herein comprise an ionizable cationic lipid. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the cationic lipid carrier has a molar ratio of 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 is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid nanoparticles have a molar ratio of about 50:38.5:10:1.5 of cationic lipid:cholesterol:PEG2000-DMG:DSPC. In some embodiments, the cationic lipid carrier has an average diameter of about 50 nanometers (nm) to about 200 nm. In some embodiments, the cationic lipid nanoparticles have an average diameter of about 80 nm to 100 nm. The compositions provided herein may comprise RNA at about 2 mg / mL or less and lipid at about 30 to about 50 mg / mL.

[0081] In some embodiments, the lipid carriers provided herein include cholesterol. In some embodiments, the lipid carriers provided herein include PEGylated lipids. In some embodiments, the lipid carriers provided herein include distearoylphosphatidylcholine (DSPC). In some embodiments, the lipid carriers provided herein include ionizable cationic lipids, cholesterol, PEGylated lipids, and distearoylphosphatidylcholine (DSPC).

[0082] The lipid nanoparticle (LNP) formulation may further include a coating with a surfactant or a polymer to improve delivery of the nanoparticles. In some embodiments, the nanoparticles may be coated with a hydrophilic coating such as, but not limited to, a PEG coating and / or a coating having a neutral surface charge. The hydrophilic coating may help deliver nanoparticles with a larger payload. In some embodiments, the lipid nanoparticles include hydrophilic polymer particles.

[0083] The nanoparticle formulations provided herein may further include carbohydrate nanoparticles comprising a carbohydrate carrier, as well as RNA encoding an antigen provided herein and / or an RNA adjuvant. Non-limiting examples of carbohydrate carriers can include, but are not limited to, succinate-modified phytoglycogen or glycogen-type materials, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, succinate-modified phytoglycogen beta-dextrin.

[0084] The lipid nanoparticles may further comprise a polymeric material (i.e., a polymer core) and / or a polymer-vitamin conjugate and / or a triblock copolymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, poly(styrene), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and / or biocompatible. The polymeric material may additionally be irradiated. By way of non-limiting example, the polymeric material may be gamma irradiated. Non-limiting examples of specific polymers include poly(caprolactone) (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-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(ester amide), polyamide, poly(ester ether), polycarbonate, polyalkylene such as polyethylene and polypropylene, poly(ethylene glycol) (PEG), polyalkylene glycol such as polyalkylene oxide (PEO), polyalkylene terephthalate such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester such as poly(vinyl acetate), polyhalogenated vinyl such as poly(vinyl chloride) (PVC), polyvinyl pyrrolidone, polysiloxane, polystyrene (PS), polyurethane, alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, derivatives of cellulose such as hydroxypropyl cellulose, carboxymethyl cellulose, poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)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 polymers of acrylic acid such as their copolymers and mixtures, polydioxanone and its copolymers, polyhydroxyalkanoate, polypropylene fumarate, polyoxymethylene, poloxamer, poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinyl pyrrolidone are mentioned.,

[0085] Lipid nanoparticles can include, but are not limited to, polynucleotides, anionic proteins (such as bovine serum albumin), surfactants (such as dimethyldioctadecylammonium bromide, for example, cationic surfactants), sugars or sugar derivatives (such as cyclodextrin), nucleic acids, polymers (such as heparin, polyethylene glycol, and poloxamer), mucolytics (such as N-acetylcysteine, mulberry, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocysteine, epratrizone, mesna, ambroxol, sobrerol, domiodol, resten, steproline, tiopronin, gelsolin, thymosin β4, dornase alfa, nertenexin, erdostein), and various surface modifiers such as various DNases including rhDNase. The surface modifier may be embedded or complexed in the surface of the particle, or may be disposed on the surface of the lipid nanoparticle (for example, by coating, adsorption, covalent bonding, or other processes).

[0086] In some embodiments, the carrier is formulated as a lipoplex including, but not limited to, the ATUPLEX™ system, DACC system, DBTC system, and other siRNA-lipoplex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.), and polyethyleneimine (PEI) or protamine-based targeted and non-targeted nucleic acid delivery.

[0087] In some embodiments, the compositions provided herein are formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLN) can be spherical with an average diameter of 10 to 1000 nm. SLN have a solid lipid core matrix that can solubilize 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 delivery of RNA to the target site that induces an immune response in the subject.

[0088] (4) Yeast cell composition As used herein, there is provided a composition comprising (a) a yeast cell comprising a permeable cell wall, and (b) one or more nucleic acids (e.g., RNA adjuvant) encoding at least a 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., RNA adjuvant) are encapsulated within the yeast cell. In some embodiments, the one or more nucleic acids (e.g., RNA adjuvant) are complexed with the yeast cell.

[0089] To prepare a composition comprising yeast cells as a delivery vehicle or carrier, the yeast cells can be contacted with a permeabilizing agent prior to complexation or encapsulation of the nucleic acids (e.g., RNA adjuvant encoding a TLR agonist) provided herein. In some embodiments, the permeabilizing agent is beta-glucanase. In some embodiments, the beta-glucanase is b-1-3-glucanase. In some embodiments, the yeast cells are Pichia pastoris cells, Saccharomyces cerevisiae cells, or Kluyveromyces lactis cells.

[0090] In some embodiments, the compositions provided herein further comprise a nucleic acid (e.g., an RNA sequence) encoding an antigen provided herein (e.g., a viral antigen, e.g., the SARS-CoV-2 spike protein or a fragment thereof). In some embodiments, the nucleic acids provided herein comprise a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein comprise a sequence that is at least 90% identical to any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein comprise a sequence that is at least 95% identical to any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein comprise any one of SEQ ID NOs: 2-175. In some embodiments, the nucleic acids provided herein encode an amino acid sequence selected from any one of SEQ ID NOs: 170-210. In some embodiments, the compositions provided herein further comprise enteric-coated microspheres. In some embodiments, the yeast cells are encapsulated within enteric-coated microspheres.

[0091] In some embodiments, the compositions provided herein are formulated for oral administration to a subject. In some embodiments, the compositions comprising the yeast cells provided herein are admixed with a food composition. In some embodiments, when the food composition is prepared or formulated as a functional food (e.g., a dietary supplement). In other embodiments, such compositions can be prepared or formulated, for example, as pharmaceuticals, health supplements, and / or medical foods.

[0092] (5) Pharmaceutical Vaccine Compositions, Dosage, and Administration Provided herein are pharmaceutical compositions and vaccine compositions comprising one or more nucleic acids provided herein or vectors provided herein and a pharmaceutically acceptable diluent, carrier, or excipient. Further provided herein are pharmaceutical compositions or vaccine compositions comprising two or more of (a) a nucleic acid encoding a TRL agonist (e.g., an RNA adjuvant), (b) a nucleic acid sequence encoding an antigen (e.g., an RNA sequence), and / or (c) a carrier, and (d) a pharmaceutically acceptable diluent, carrier, or excipient. Further provided herein are pharmaceutical compositions comprising a vector that comprises or encodes any one of the nucleic acids (e.g., an RNA adjuvant) or proteins provided herein and a pharmaceutically acceptable diluent, carrier, or excipient.

[0093] In some embodiments, the compositions provided herein (e.g., an RNA adjuvant) are combined with a pharmaceutically acceptable salt, excipient, and / or carrier to form a pharmaceutical composition. The pharmaceutical salts, excipients, and carriers may be selected based on the route of administration, the location of the target issue, and the time course of drug delivery. Pharmaceutically acceptable carriers or excipients may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration.

[0094] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid, or gas (aerosol). Injectable preparations, for example, sterile aqueous or oily suspension injections, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injection solution, suspension injection, or injectable emulsion in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that can be used include water, Ringer's solution (U.S.P.), and isotonic sodium chloride solution. In addition, sterile non-volatile oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating non-volatile oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0095] RNA adjuvants, nucleic acids, compositions, vaccine compositions, and pharmaceutical compositions for administration to a subject in need can be formulated in unit dosage forms for ease of administration and uniformity of dosage. A unit dosage form is a physically discrete unit of the compositions provided herein that is appropriate for the subject to be treated. However, it will be understood that the total usage of the compositions provided herein will be determined by the attending physician within the scope of sound medical judgment. For any of the compositions provided herein, a therapeutically effective dosage can first be estimated either 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 desired concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration in humans. The therapeutic efficacy and toxicity of the compositions provided herein, e.g., ED 50 (wherein the dosage is therapeutically effective in 50% of the population) and LD 50(When such a dosage is lethal to 50% of a population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio of LD 50 / ED 50 . Pharmaceutical compositions showing a large therapeutic index may be useful in some embodiments. Data obtained from cell culture assays and animal studies may be used in formulating dosage ranges for human use.

[0096] Provided herein are nucleic acids, RNA adjuvants, compositions, vaccine compositions, and pharmaceutical compositions for administration to a subject in need thereof (e.g., as a vaccine or as a therapeutic agent for an infectious disease or cancer). In some embodiments, administration of the compositions provided herein is by topical administration. In some embodiments, administration of the compositions provided herein is by systemic administration. In some embodiments, the compositions provided herein are formulated for administration / use in administration via subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, or oral routes. In some cases, the treatment regimen is given according to the weight of the subject. For example, body mass index may be used. BMI is calculated by BMI = weight (kg) / [height (m)] 2 .

[0097] Although merely exemplary, exemplary dosages for the nucleic acids and RNA vaccines provided herein are provided below. In some embodiments, the nucleic acid encoding an RNA adjuvant or TLR agonist provided herein and / or the nucleic acid encoding an antigen 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 a maximum of 500 ng. In some embodiments, the nucleic acids provided herein are 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 acids provided herein are 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 a maximum of 100 μg.

[0098] In some embodiments, the compositions provided herein are administered intravenously or intramuscularly. In some embodiments, the compositions provided herein are administered at a concentration 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 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 about 2500 mg / kg.

[0099] In some embodiments, the compositions provided herein are administered orally. In some embodiments, the compositions provided herein are administered orally at a concentration 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 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 about 2500 mg / kg.

[0100] In some embodiments, the dosage of the compositions provided herein can be administered once daily or divided into sub-dosages and administered in multiple dosages, e.g., daily, twice daily, weekly, biweekly, monthly, bimonthly, annually, etc. In some embodiments, the administration is repeated at least about once every year (8760 hours, 365 days), every two years, every three years, every four years, every five years, or at longer intervals. In some embodiments, additional dosages of the nucleic acid, pharmaceutical composition, or vaccine composition are administered to the subject. In some embodiments, the administration includes administration of a first dosage of the pharmaceutical composition and administration of a second dosage of the pharmaceutical composition at least about two weeks after the first dosage. In some embodiments, the composition is a booster vaccine composition that is the second, third, or fourth dosage of the composition administered to the subject.

[0101] RNA adjuvants and RNA vaccine compositions (e.g., RNA encoding an antigen) can be administered in the same composition, in parallel, or sequentially. The RNA encoding the TLR agonist provided herein can be on the same nucleic acid strand or a different nucleic acid strand from the RNA sequence encoding the antigen. When administered sequentially, the dosing interval is selected to be beneficial, inter alia, to the therapeutic efficacy and / or safety of the combination therapy. In certain embodiments, the RNA adjuvant composition provided herein can be administered first, followed by administration of the RNA vaccine, or alternatively, the RNA vaccine can be administered first, followed by administration of the RNA adjuvant composition of the present disclosure (e.g., a composition comprising the RNA sequences listed in Table 1). By way of non-limiting example, the dosing interval can be about 1 hour (hr), about 2 hours (hrs), about 4 hours, about 6 hours, about 12 hours, about 16 hours, or about 20 hours. In certain embodiments, the dosing interval 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 days. In some embodiments, the dosing interval is about 1 week (7 days), 2 weeks, 3 weeks, or 4 weeks, or longer. In some embodiments, the dosing interval is about 1 month (30 days), 2 months (60 days), or longer. When administered in parallel, the RNA adjuvant composition can be administered separately by the same or different routes simultaneously with the second RNA vaccine, or administered by the same route in a single pharmaceutical composition. In certain embodiments, the amount and frequency of administration of the RNA vaccine can use the standard dosage and standard frequency of administration used for that particular RNA vaccine. In some embodiments, the dosage of the RNA vaccine is reduced when administered with the RNA adjuvant composition provided herein. This is due to the enhanced effect of the RNA adjuvant-RNA vaccine combination on the innate immune response.

[0102] (6) Therapeutic Use The compositions and adjuvants provided herein are formulated for use in the treatment of a disease or condition. Also provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject an agent in combination with an adjuvant provided herein. In some embodiments, the disease or disorder is cancer or an infectious disease. 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 agent, or an immunotherapy) in the same composition or in separate compositions.

[0103] Also provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject an agent in combination with an adjuvant provided herein. In some embodiments, the agent is administered prior to administration of the adjuvant. In some embodiments, the agent is administered concurrently with the adjuvant. In some embodiments, the agent is administered after administration of the adjuvant. In some embodiments, the compositions provided herein comprise an agent and an adjuvant in a unit dosage form.

[0104] In some embodiments, administration of an effective amount of an agent or composition provided herein to a subject in the presence of an antigen is followed by induction of a first innate immune response in the subject. In some embodiments, the innate immune response comprises an increase in the level or activity of nuclear factor kappa B (NF-κB) in the epithelial cells of the subject as compared to the level or activity of NF-κB in the absence of the composition.

[0105] In some embodiments, the subject has an infectious disease, has been diagnosed with an infectious disease, or is at risk of developing an infectious disease. In some embodiments, the infectious disease is a viral infectious disease. In some embodiments, the viral infectious disease is an upper respiratory viral infectious disease. In some embodiments, the upper respiratory viral infectious disease is COVID-19, SARS, MERS, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia, or rhinovirus infection. In some embodiments, the infectious disease is a bacterial infectious disease, a fungal infectious disease, a parasitic infectious disease, or a yeast infectious disease. In some embodiments, the subject has contracted an infectious disease through contact with another infected subject. In some embodiments, the subject has contracted an infectious disease from a different species that harbors the microorganism. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0106] Provided herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject a composition provided herein in an amount sufficient to stimulate an immune response.

[0107] Provided herein is a method of enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, the method comprising: (a) administering to the 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 comprising RNA encoding the antigen, whereby the immune response against the antigen encoded by the RNA is enhanced.

[0108] 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 derived from the SARS-CoV-2 virus. In some embodiments, the composition induces an immune response against a viral antigen, and the viral antigen is selected from adenovirus, astrovirus, bocavirus, bunyavirus, BK virus, coltivirus, 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, norovirus, 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.

[0109] The present specification further provides a method for treating cancer in a subject, the method comprising administering a composition provided herein, whereby the cancer is treated. In some embodiments, the subject has cancer, is diagnosed with cancer, or is at risk of developing cancer. In some embodiments, the administration is intratumoral, intrasplenic, intradermal, intra-articular, intramuscular, oral, intranasal, or intravenous. In some embodiments, the subject has a solid tumor or a hematological cancer. In some embodiments, the subject has 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, or is suspected of having or is diagnosed with having the same. In some embodiments, the subject has a solid tumor. In some embodiments, the subject has metastatic cancer. In some embodiments, the composition for use in treating cancer comprises RNA or DNA encoding a tumor antigen provided herein. In some embodiments, the composition for use in treating cancer comprises RNA encoding an immunomodulatory protein provided herein.

[0110] Exemplary embodiments This specification provides a composition for inducing an immune response following administration to a subject, the composition comprising: (a) an RNA adjuvant encoding a protein, the protein comprising at least a functional fragment of a toll-like receptor 5 (TLR5) agonist, the functional fragment activating the TLR5 pathway in a cell upon contact with the cell; and (b) one or more delivery vehicles, wherein administration of an effective amount of the composition to a subject in the presence of an antigen induces a first innate immune response in the subject. This specification further provides a composition wherein one or more delivery vehicles are formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration. This specification further provides a composition wherein a functional fragment of a TLR5 agonist activates the TLR5 pathway in a cell upon contact with the cell. This specification further provides a composition wherein the cell is an epithelial cell. This specification further provides a composition wherein a functional fragment of a TLR5 agonist comprises a functional derivative of a bacterial motility protein. This specification further provides a composition wherein the bacterial motility protein is a bacterial flagellin protein, a derivative thereof, or a functional fragment thereof. This specification further provides a composition wherein the protein is a truncated form of a bacterial flagellin protein, a secreted form of a bacterial flagellin protein, or a combination thereof. This specification further provides a composition wherein the functional fragment of the toll-like receptor 5 (TLR5) agonist comprises the D0 and D1 domains of a bacterial flagellin protein. This specification further provides a composition wherein the functional fragment of the toll-like receptor 5 (TLR5) agonist comprises the D0, D1, and D2 domains of a bacterial flagellin protein. This specification further provides a composition wherein the functional fragment of the toll-like receptor 5 (TLR5) agonist comprises the D0, D1, D2, and D3 domains of a bacterial flagellin protein. This specification further provides a composition wherein the synthetic protein further comprises a signal sequence. This specification further provides a composition wherein the signal sequence comprises the amino acid sequence of MRSLSVLALLLLLLLAPASA (SEQ ID NO: 1).Also provided herein are compositions wherein the RNA adjuvant comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions for inducing an immune response wherein the RNA adjuvant comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions wherein the RNA adjuvant comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions wherein the RNA adjuvant comprises a sequence that is at least 96% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions wherein the RNA adjuvant comprises a sequence that is at least 97% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions wherein the RNA adjuvant comprises a sequence that is at least 98% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions wherein the RNA adjuvant comprises a sequence that is at least 99% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NOs: 2-37. 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, a vector, or any combination thereof. Also provided herein are compositions wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen. Also provided herein are compositions wherein the antigen is a tumor antigen. Also provided herein are compositions wherein the composition further comprises RNA encoding the antigen, and the RNA encoding the antigen is within the same delivery vehicle as the RNA adjuvant. Also provided herein are compositions wherein the composition further comprises RNA encoding the antigen, and the RNA encoding the antigen is within a delivery vehicle different from the RNA adjuvant. Also provided herein are compositions wherein the RNA adjuvant further comprises the RNA sequence encoding the antigen.Also provided herein are compositions in which an RNA sequence encoding an antigen is operably linked to an RNA adjuvant. Also provided herein are compositions in which the innate immune response comprises an increase in the level or activity of nuclear factor kappa B (NF-κB) in the epithelial cells of a subject compared to the level or activity of NF-κB in the absence of the composition.

[0111] Also provided herein are compositions for inducing an immune response following administration to a subject, the compositions comprising: (a) a nucleic acid sequence encoding a protein, the protein comprising at least a functional fragment of a toll-like receptor 5 (TLR5) agonist, the functional fragment activating the TLR5 pathway in a cell upon contact with the cell; and (b) one or more delivery vehicles, wherein administration of an effective amount of the composition to the subject in the presence of an antigen induces a first innate immune response in the subject. Also provided herein are compositions in which the nucleic acid sequence comprises an RNA sequence or a DNA sequence. Also provided herein are compositions in which the protein is a synthetic protein.

[0112] As used herein, there is provided a composition comprising: (a) a lipid carrier; and (b) an RNA sequence encoding at least a functional fragment of a bacterial motility protein, wherein the RNA adjuvant is a TLR5 agonist. Further provided herein is a composition wherein the RNA adjuvant is encapsulated within the lipid carrier. Further provided herein is a composition wherein the RNA adjuvant is complexed with the lipid carrier. Further provided herein is a composition wherein the lipid carrier is a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polyethylene glycol (PEG) lipid carrier, a polymeric lipid nanoparticle, a polymeric conjugate lipid carrier, a synthetic vesicle, a liposome, an exosome, an endosome, a lipoplex, a lipidoid, derivatives thereof, variants, or any combination thereof. Further provided herein is a composition wherein at least a functional fragment of the bacterial motility protein is a bacterial flagellin protein, a derivative thereof, or a functional fragment. Further provided herein is a composition wherein the bacterial motility protein is a truncated form of a bacterial flagellin protein, a secreted form of a bacterial flagellin protein, or a combination thereof. Further provided herein is a composition wherein the functional fragment of the bacterial motility protein comprises the D0 and D1 domains of the bacterial flagellin protein. Further provided herein is a composition wherein the functional fragment of the bacterial motility protein comprises the D0, D1, and D2 domains of the bacterial flagellin protein. Further provided herein is a composition wherein the functional fragment of the bacterial motility protein comprises the D0, D1, D2, and D3 domains of the bacterial flagellin protein. Further provided herein is a composition wherein the RNA adjuvant comprises mRNA. Further provided herein is a composition wherein the composition further comprises one or more RNA molecules each encoding an antigen. Further provided herein is a composition wherein the composition further comprises one or more antigens. Further provided herein is a composition wherein the RNA encoding the antigen is mRNA. Further provided herein is a composition wherein the mRNA is chemically modified.Also provided herein are compositions in which the chemical modification comprises a poly-A tail, a chemically modified nucleobase, or a 5' end cap. Also provided herein are compositions in which the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen. Also provided herein are compositions in which the viral antigen is derived from adenovirus, astrovirus, bocavirus, bunyavirus, BK virus, coltivirus, 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. Also provided herein are compositions in which the SARS virus is the SARS-CoV-2 virus. Also provided herein are compositions in which the antigen comprises a spike protein, a glycoprotein, or a hemagglutinin protein. Also provided herein are compositions in which the RNA encoding the antigen is encapsulated within the same lipid carrier as the RNA adjuvant. Also provided herein are compositions in which the RNA encoding the antigen is encapsulated within a lipid carrier different from the RNA adjuvant. Also provided herein are compositions in which the RNA adjuvant comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions in which the RNA adjuvant comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 2-37. Also provided herein are compositions in which the RNA adjuvant comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 2-37.The present specification further provides a composition, wherein the RNA adjuvant comprises a sequence selected from any one of SEQ ID NOs: 2 to 37. The present specification further provides a composition in the form of a suspension, an aqueous solution, or an emulsion.

[0113] The present specification provides a composition comprising (a) a carrier and (b) a nucleic acid encoding at least a functional fragment of a bacterial motility protein, wherein the functional fragment of the bacterial motility protein is a TLR5 agonist. The present specification further provides a composition, wherein the carrier is a lipid carrier. The present specification further provides a composition, wherein the nucleic acid comprises RNA, DNA, or a combination thereof.

[0114] Provided herein is a composition for inducing an immune response following administration to a subject, the composition comprising: (a) an RNA adjuvant encoding a protein, the protein comprising at least a functional fragment of a toll-like receptor 4 (TLR4) agonist; and (b) one or more delivery vehicles formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration, wherein administration of an effective amount of the composition to a subject in the presence of an antigen induces a first innate immune response in the subject. Further provided herein is a composition for inducing an immune response, wherein the TLR4 agonist comprises at least a functional fragment of a high mobility group box 1 (HMGB1) protein, nicotinic acid nucleotide--dimethylbenzimidazole phosphoribosyltransferase, transglycosylase, 50S ribosomal protein, heparin-binding hemagglutinin protein, YadA family autotransporter adhesin, dnaJ protein, pneumolysin protein, Omp19 protein, 6,7-dimethyl-8-ribityllumazine synthase, or any combination thereof. The TLR4 agonist is of bacterial origin. Further provided herein is a composition for inducing an immune response, wherein the TLR4 agonist is of human origin. Further provided herein is a composition for inducing an immune response, wherein the RNA comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 38-103. Further provided herein is a composition for inducing an immune response, wherein the RNA comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 38-103. Further provided herein is a composition for inducing an immune response, wherein the RNA comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 38-103. Further provided herein is a composition for inducing an immune response, wherein the RNA comprises a sequence selected from any one of SEQ ID NOs: 38-103. Further provided herein is a composition for inducing an immune response, wherein the delivery vehicle is a diluent, aqueous solution, particle, emulsion, suspension, lipid carrier, vesicle, or any combination thereof.Also provided herein is a composition for inducing an immune response, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen. Also provided herein is a composition for inducing an immune response, wherein the innate immune response comprises an increase in the level or activity of nuclear factor kappa B upon administration of the composition as compared to the level or activity of NF-kB in the absence of the composition.

[0115] As used herein, there is provided a composition comprising: (a) a lipid carrier; and (b) an RNA sequence encoding at least a functional fragment of a TLR4 agonist. Further provided herein is a composition wherein the TLR4 agonist comprises at least a functional fragment of a high mobility group box 1 (HMGB1) protein, nicotinic acid nucleotide-dimethylbenzimidazole phosphoribosyltransferase, transglycosylase, 50S ribosomal protein, heparin-binding hemagglutinin protein, YadA family autotransporter adhesin, dnaJ protein, pneumolysin protein, Omp19 protein, 6,7-dimethyl-8-ribityllumazine synthase, or any combination thereof. Further provided herein is a composition wherein the TLR4 agonist is of bacterial origin. Further provided herein is a composition wherein the TLR4 agonist is of human origin. Further provided herein is a composition wherein the RNA comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 38-103. Further provided herein is a composition wherein the RNA comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 38-103. Further provided herein is a composition wherein the RNA comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 38-103. Further provided herein is a composition wherein the RNA comprises a sequence selected from any one of SEQ ID NOs: 38-103. Further provided herein is a composition wherein the RNA adjuvant is encapsulated within the lipid carrier. Further provided herein is a composition wherein the RNA adjuvant is complexed with the lipid carrier. Further provided herein is a composition 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 lipoplex, a lipidoid, derivatives or variants thereof, or any combination thereof. Further provided herein is a composition wherein the RNA adjuvant comprises mRNA.Also provided herein are compositions that further comprise one or more RNA molecules each encoding an antigen. Also provided herein are compositions that further comprise one or more antigens. Also provided herein are compositions wherein the RNA encoding the antigen is mRNA. Also provided herein are compositions 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. Also provided herein are compositions wherein the viral antigen is derived from adenovirus, astrovirus, bocavirus, bunyavirus, BK virus, coltivirus, 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. Also provided herein are compositions wherein the SARS virus is the SARS-CoV-2 virus. Also provided herein are compositions wherein the antigen comprises a spike protein, a glycoprotein, a hemagglutinin protein, or a cancer cell protein. Also provided herein are compositions wherein the RNA encoding the antigen is encapsulated within the same lipid carrier as the RNA adjuvant. Also provided herein are compositions wherein the RNA encoding the antigen is encapsulated within a lipid carrier different from the RNA adjuvant. Also provided herein are compositions wherein the composition is in the form of a suspension, an aqueous solution, or an emulsion.

[0116] As used herein, there is provided a composition comprising (a) a carrier and (b) a nucleic acid encoding at least a functional fragment of a TLR4 agonist. Further provided herein is a composition wherein the carrier is a lipid carrier. Further provided herein is a composition wherein the nucleic acid comprises RNA, DNA, or a combination thereof.

[0117] The present specification provides a composition for inducing an immune response following administration to a subject, the composition comprising: (a) an RNA adjuvant encoding a protein, the protein comprising at least a functional fragment of a toll-like receptor 2 (TLR2) agonist; and (b) one or more delivery vehicles, wherein administration of an effective amount of the composition to a subject in the presence of an antigen induces a first innate immune response in the subject. The present specification further provides a composition wherein one or more delivery vehicles are formulated for intranasal administration, oral administration, intradermal administration, intra-articular administration, intra-splenic administration, intratumoral administration, intravenous administration, or intramuscular administration. The present specification further provides a composition wherein the TLR2 agonist comprises an outer membrane protein (Omp), a Panton-Valentine leukocidin (PVL) protein, a polyprotein, a TRAP transporter protein, a hemagglutinin protein, an oxidoreductase protein, or at least a functional fragment of a type VII secretion system. The present specification further provides a composition wherein the TLR2 agonist is derived from bacteria. The present specification further provides a composition wherein the RNA adjuvant comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 104 to 175. The present specification further provides a composition wherein the RNA adjuvant comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 104 to 175. The present specification further provides a composition wherein the RNA adjuvant comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 104 to 175. The present specification further provides a composition wherein the RNA comprises a sequence selected from any one of SEQ ID NOs: 104 to 175. The present specification further provides a composition wherein one or more delivery vehicles comprise a diluent, an aqueous solution, particles, an emulsion, a suspension, a lipid carrier, vesicles, or any combination thereof. The present specification further provides a composition wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen. The present specification further provides a composition wherein the innate immune response comprises an increase in the level or activity of nuclear factor kappa B in the subject at the time of administration of the composition compared to the level or activity of NF-kB in the absence of administration of the composition.

[0118] In this specification, a composition is provided that comprises (a) a lipid carrier and (b) an RNA sequence encoding at least a functional fragment of a TLR2 agonist. Further provided in this specification is a composition wherein the TLR2 agonist comprises an outer membrane protein (Omp), a Panton-Valentine bicomponent leukocidin (PVL) protein, a polymorphic protein, a TRAP transporter protein, a hemagglutinin protein, an oxidoreductase protein, or at least a functional fragment of a type VII secretion system. Further provided in this specification is a composition wherein the TLR2 agonist is derived from bacteria. Further provided in this specification is a composition wherein the RNA sequence comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 104 to 175. Further provided in this specification is a composition wherein the RNA sequence comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 104 to 175. Further provided in this specification is a composition wherein the RNA sequence comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 104 to 175. Further provided in this specification is a composition wherein the RNA sequence comprises a sequence selected from any one of SEQ ID NOs: 104 to 175. Further provided in this specification is a composition wherein the RNA sequence is encapsulated within the lipid carrier. Further provided in this specification is a composition wherein the RNA sequence is complexed with the lipid carrier. Further provided in this specification is a composition 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 lipoplex, a lipidoid, derivatives thereof, variants, or any combination thereof. Further provided in this specification is a composition wherein the RNA sequence comprises mRNA. Further provided in this specification is a composition wherein the composition further comprises one or more nucleic acids each encoding an antigen. Further provided in this specification is a composition wherein the composition further comprises one or more antigens. Further provided in this specification is a composition wherein the RNA encoding the antigen is mRNA. Further provided in this specification is a composition wherein the mRNA is chemically modified.Also provided herein are compositions in which the chemical modification comprises a poly-A tail, a chemically modified nucleobase, or a 5′ end cap. Also provided herein are compositions in which the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen. Also provided herein are compositions in which the viral antigen is derived from adenovirus, astrovirus, bocavirus, bunyavirus, BK virus, coltivirus, 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. Also provided herein are compositions in which the SARS virus is the SARS-CoV-2 virus. Also provided herein are compositions in which the antigen comprises a spike protein, a glycoprotein, a hemagglutinin protein, or a cancer cell protein. Also provided herein are compositions in which the RNA sequence encoding the antigen is encapsulated within the same lipid carrier as the RNA sequence. Also provided herein are compositions in which the RNA sequence encoding the antigen is encapsulated within a lipid carrier different from the RNA sequence encoding a TLR2 agonist. Also provided herein are compositions in which the composition is in the form of a suspension, an aqueous solution, or an emulsion.

[0119] As used herein, there is provided a composition comprising: (a) a carrier; and (b) a nucleic acid encoding at least a functional fragment of a TLR2 agonist. Further provided herein is a composition wherein the carrier is a lipid carrier. Further provided herein is a composition wherein the nucleic acid comprises RNA, DNA, or a combination thereof.

[0120] This specification provides a composition for inducing an immune response upon administration to a subject, the composition comprising: (a) one or more carriers; (b) one or more nucleic acids encoding (i) at least a 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. Following administration of an effective amount of the composition, a innate immune response is induced in the subject in the presence of the viral antigen, thereby inducing an immune response against the viral antigen. This specification further provides a composition wherein the one or more nucleic acids comprise RNA, DNA, or a combination thereof. This specification further provides a composition wherein the composition comprises an RNA sequence encoding a functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or any combination thereof. This specification further provides a composition wherein the composition comprises RNA encoding a viral antigen. This specification further provides a composition wherein the one or more carriers comprise a lipid carrier. This specification further provides a composition wherein the delivery vehicle is formulated for intranasal, oral, intradermal, intra-articular, intrasplenic, intratumoral, intravenous, or intramuscular administration. This specification further provides a composition wherein a functional fragment of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or any combination thereof activates the TLR4, TLR2, or TLR5 pathway in a cell upon contact with the cell. This specification further provides a composition wherein the cell is an epithelial cell. This specification further provides a composition wherein the viral antigen is a spike protein, a glycoprotein, or a viral envelope protein. This specification further provides a composition wherein the spike protein is derived from the SARS-CoV-2 virus. This specification further provides a composition wherein the RNA encoding the viral antigen is mRNA. This specification further provides a composition wherein the RNA encoding the viral antigen is chemically modified.Also provided herein are compositions in which the chemical modification comprises a poly-A tail, a chemically modified nucleobase, or a 5’-end cap. Also provided herein are compositions in which one or more carriers are complexed with one or more RNA adjuvants. Also provided herein are compositions in which one or more carriers are complexed with one or more nucleic acids encoding a viral antigen. Also provided herein are compositions in which one or more nucleic acids are encapsulated by one or more lipid carriers. Also provided herein are compositions in which one or more nucleic acids encoding a viral antigen are encapsulated within one or more carriers. Also provided herein are compositions in which 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. Also provided herein are compositions in which one or more nucleic acids comprise a sequence selected from SEQ ID NOs: 2-175, or a functional fragment thereof. Also provided herein are compositions in which administration of the composition to a subject induces a innate immune response in the subject that is greater than the innate immune response in a subject not administered an effective amount of the composition. Also provided herein are compositions in which administration of the composition to a subject induces a innate immune response in the subject that is greater than the innate immune response in a subject administered only a composition comprising a nucleic acid encoding a viral antigen alone. Also provided herein are compositions in which the innate immune response comprises an increase in the level or activity of nuclear factor kappa B upon administration of an effective amount of the composition compared to the level or activity of NF-kB in the absence of the composition.

[0121] Also provided herein are compositions comprising (a) an RNA adjuvant comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2-175, and (b) a delivery vehicle. Also provided herein are compositions in which 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.

[0122] As used herein, a composition is provided that comprises (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. Further provided herein is a composition 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.

[0123] As used herein, a composition is provided that comprises (a) a protein comprising an amino acid sequence that is 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% identical to a sequence selected from any one of SEQ ID NOs: 176 to 217, and (b) a delivery vehicle. Further provided herein is a composition 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.

[0124] As used herein, a composition is provided that 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. Further provided herein is a composition 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.

[0125] As used herein, a composition is provided that 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. Further provided herein is a composition 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. Further provided herein is a composition that comprises one or more nucleic acids, wherein the one or more nucleic acids comprise RNA, DNA, or a combination thereof. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 80% identical to SEQ ID NO: 206. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 80% identical to SEQ ID NO: 210. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 212. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 85% identical to SEQ ID NO: 213. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 215. Further provided herein is a nucleic acid encoding an amino acid sequence that is at least 75% identical to SEQ ID NO: 217.

[0126] As used herein, there is provided a composition comprising a synthetic protein comprising a protein construct, wherein the protein construct comprises an amino acid sequence selected from SEQ ID NOs: 176 to 217. Further provided herein is a composition comprising two or more, three or more, four or more, or five or more protein constructs. Further provided herein is a nucleic acid encoding a protein construct that is at least 80% identical to SEQ ID NO: 206. Further provided herein is a nucleic acid encoding a protein construct that is at least 80% identical to SEQ ID NO: 210. Further provided herein is a nucleic acid encoding a protein construct that is at least 75% identical to SEQ ID NO: 212. Further provided herein is a nucleic acid encoding a protein construct that is at least 85% identical to SEQ ID NO: 213. Further provided herein is a nucleic acid encoding a protein construct that is at least 75% identical to SEQ ID NO: 215. Further provided herein is a nucleic acid encoding a protein construct that is at least 75% identical to SEQ ID NO: 217.

[0127] As used herein, a composition is provided that comprises: (a) yeast cells having a permeable cell wall, and (b) one or more RNA adjuvants that are at least functional fragments of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. As used herein, a composition is provided that comprises: (a) yeast cells having a permeable cell wall, and (b) one or more nucleic acids that encode at least functional fragments of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. As used herein, a composition is provided that comprises: (a) yeast cells having a permeable cell wall, and (b) one or more synthetic proteins that comprise at least functional fragments of a TLR4 agonist, a TLR2 agonist, a TLR5 agonist, or a combination thereof. Further provided herein is a composition that further comprises RNA encoding a viral antigen. Further provided herein is a composition that further comprises DNA encoding a viral antigen. Further provided herein is a composition that further comprises chemically modified RNA encoding a viral antigen. Further provided herein is a composition wherein the one or more RNA adjuvants or the nucleic acid comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 2 to 175. Further provided herein is a composition wherein the one or more RNA adjuvants or the nucleic acid comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 2 to 175. Further provided herein is a composition wherein the one or more RNA adjuvants or the nucleic acid comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 to 175. Further provided herein is a composition wherein the one or more RNA adjuvants or the nucleic acid comprises any one of SEQ ID NOs: 2 to 175.Also provided herein are compositions wherein one or more RNA adjuvants or the nucleic acid encodes an amino acid sequence that is 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% identical to a sequence selected from any one of SEQ ID NOs: 176 to 217. Also provided herein are nucleic acids that encode an amino acid sequence that is at least 80% identical to SEQ ID NO: 206. Also provided herein are nucleic acids that encode an amino acid sequence that is at least 80% identical to SEQ ID NO: 210. Also provided herein are nucleic acids that encode an amino acid sequence that is at least 75% identical to SEQ ID NO: 212. Also provided herein are nucleic acids that encode an amino acid sequence that is at least 85% identical to SEQ ID NO: 213. Also provided herein are nucleic acids that encode an amino acid sequence that is at least 75% identical to SEQ ID NO: 215. Also provided herein are nucleic acids that encode an amino acid sequence that is at least 75% identical to SEQ ID NO: 217. Also provided herein are compositions wherein one or more RNA adjuvants or the nucleic acid are encapsulated within yeast cells. Also provided herein are compositions wherein the composition further comprises enteric-coated microspheres and the yeast cells are encapsulated within the enteric-coated microspheres. Also provided herein are compositions wherein the composition is formulated for oral administration to a subject.

[0128] Provided herein is a composition for inducing an immune response upon administration to a subject, the composition comprising: (a) one or more carriers; (b) one or more nucleic acids encoding (i) at least a functional fragment of a TLR4 agonist, TLR2 agonist, 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. Following administration of an effective amount of the composition, a innate immune response is induced in the subject in the presence of the tumor antigen, thereby inducing an immune response against the tumor antigen. Further provided herein is a composition wherein the one or more carriers comprise a lipid carrier. Further provided herein is a composition wherein the immunomodulatory protein is CD83, 4-1BB ligand, or a cytokine. Further provided herein is a composition 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), preferentially expressed antigen in melanoma (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. Further provided herein is a composition wherein the one or more nucleic acids comprise RNA, DNA, or a combination thereof. Further provided herein is a composition wherein the one or more nucleic acids encoding the tumor antigen and / or the immunomodulatory protein are chemically modified. Further provided herein is a composition wherein the chemical modification comprises a poly-A tail, a chemically modified nucleobase, or a 5' end cap. Further provided herein is a composition wherein the one or more lipid carriers are complexed with one or more RNA adjuvants. Further provided herein is a composition wherein the one or more lipid carriers are complexed with at least one RNA encoding a tumor antigen and / or an immunomodulatory protein.Also provided herein are compositions in which one or more RNA adjuvants are encapsulated by one or more lipid carriers. Also provided herein are compositions in which at least one RNA encoding a tumor antigen and / or an immunomodulatory protein is encapsulated by one or more lipid carriers. Also provided herein are compositions in which one or more RNA adjuvants are operably linked to at least one RNA encoding a viral antigen, a derivative thereof, or a functional fragment. Also provided herein are compositions in which one or more RNA adjuvants comprise a sequence selected from SEQ ID NOs: 2-175, or a functional fragment thereof. Also provided herein are compositions in which the innate immune response induced in a subject is greater than the innate immune response in a subject not administered an effective amount of the composition.

[0129] The present specification provides a vector, which contains one or more nucleic acids encoding a TLR5 agonist, a TLR4 agonist, a TLR2 agonist, or a combination thereof. The present specification further provides a vector, which includes a viral vector. The present specification further provides a vector, wherein the viral vector includes 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. The present specification further provides a vector, wherein the TLR5 agonist includes a functional derivative of a bacterial motility protein. The present specification further provides a vector, wherein the TLR5 agonist includes a DNA sequence encoding an RNA sequence that is at least 85% identical to any one of SEQ ID NOs: 2 to 37. The present specification further provides a vector, wherein the TLR5 agonist includes a sequence that is at least 85% identical to any one of SEQ ID NOs: 2 to 37. The present specification further provides a vector, wherein the TLR4 agonist includes at least a functional fragment of a high-mobility group box 1 (HMGB1) protein, a nicotinic acid nucleotide--dimethylbenzimidazole phosphoribosyltransferase, a transglycosylase, a 50S ribosomal protein, a heparin-binding hemagglutinin protein, a YadA family autotransporter adhesin, a dnaJ protein, a pneumolysin protein, an Omp19 protein, a 6,7-dimethyl-8-ribityllumazine synthase, or any combination thereof. The present specification further provides a vector, wherein the one or more nucleic acids include a DNA sequence encoding an RNA sequence that is at least 85% identical to any one of SEQ ID NOs: 38 to 103. The present specification further provides a vector, wherein the one or more nucleic acids include a sequence that is at least 85% identical to any one of SEQ ID NOs: 38 to 103.Also provided herein is a vector, wherein the TLR2 agonist comprises an outer membrane protein (Omp), a Panton-Valentine leucocidin (PVL) protein, a porin protein, a TRAP transporter protein, a hemagglutinin protein, an oxidoreductase protein, or at least a functional fragment of a type VII secretion system. Also provided herein is a vector, wherein one or more nucleic acids comprise a DNA sequence encoding an RNA sequence that is at least 85% identical to any one of SEQ ID NOs: 104-175. Also provided herein is a vector, wherein one or more nucleic acids comprise a sequence that is at least 85% identical to any one of SEQ ID NOs: 104-175. Also provided herein is a vector, 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. In some embodiments, the nucleic acid sequence is an RNA sequence or a DNA sequence. Also provided herein is a vector, wherein the viral antigen comprises a SARS-CoV-2 spike protein.

[0130] Also provided herein is a composition, wherein the composition comprises a vector provided herein and a lipid carrier.

[0131] Also provided herein is a vaccine composition, wherein the vaccine composition comprises a composition provided herein and a pharmaceutically acceptable excipient.

[0132] Also provided herein is a vaccine composition, wherein the vaccine composition comprises (a) 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: 2-175, and (c) one or more RNAs encoding an antigen. Also provided herein is a vaccine composition, wherein each of the one or more RNAs encoding an antigen encodes a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or any combination thereof.

[0133] This specification provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of the composition provided herein or the vaccine composition provided herein, whereby the immune response is stimulated. This specification further provides a method for stimulating an immune response in a subject, wherein the administration is systemic or topical. This specification further provides a method for stimulating an immune response in a subject, wherein the administration is intramuscular, intravenous, oral, intradermal, intratumoral, inhalation, subcutaneous, or intraperitoneal. This specification further provides a method for stimulating an immune response in a subject, wherein the administration is daily, weekly, monthly, or annually. This specification further provides a method for stimulating an immune response in a subject, the method further comprising administering to the subject an RNA vaccine composition. This specification further provides a method for stimulating an immune response in a subject, wherein the subject has an infectious disease or is at risk of developing an infectious disease. This specification further provides a method for stimulating an immune response in a subject, wherein the infectious disease is a viral infection. This specification further provides a method for stimulating an immune response in a subject, wherein the viral infection is an upper respiratory viral infection. This specification further provides a method for stimulating an immune response in a subject, wherein the upper respiratory viral infection is COVID-19, SARS, MERS, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia, or rhinovirus infection. This specification further provides a method for stimulating an immune response in a subject, wherein the infectious disease is a bacterial infection, a fungal infection, a parasitic infection, or a yeast infection. This specification further provides 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. This specification further provides a method for stimulating an immune response in a subject, wherein the spike protein is derived from the SARS-CoV-2 virus. This specification further provides 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 the composition or the vaccine composition is administered to the subject.

[0134] The present specification provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, the method comprising: (a) administering to a subject an effective amount of the composition provided herein or the vaccine composition provided herein; and (b) administering to the subject an RNA vaccine composition comprising RNA encoding the antigen, whereby the immune response against the antigen encoded by the RNA is enhanced. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the administration is systemic administration or local administration. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumoral administration, inhalation, subcutaneous administration, or intraperitoneal administration. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the administration is daily, weekly, monthly, or yearly. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the subject has an infectious disease or is at risk of developing an infectious disease. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the infectious disease is a viral infection. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the viral infection is an upper respiratory viral infection. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the upper respiratory viral infection is COVID-19, SARS, MERS, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia, or rhinovirus infection. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the infectious disease is a bacterial infection, a fungal infection, a parasitic infection, or a yeast infection.The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the antigen is a spike protein, a glycoprotein, a viral envelope protein, or a tumor antigen. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the spike protein is derived from the SARS-CoV-2 virus. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the subject is a mammal. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the subject is a human. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein an additional dose of the composition or the vaccine composition is administered to the subject. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the subject has cancer, is diagnosed with cancer, or is at risk of developing cancer. The present specification further provides a method for enhancing an immune response against an antigen encoded by RNA in an RNA vaccine composition, wherein the subject has a solid tumor or a hematological cancer. The present specification further provides a method for enhancing an immune response against an antigen encoded by 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.

[0135] This specification provides a method for the treatment of an infectious disease in a subject, the method comprising administering to the subject an effective amount of the composition provided herein or the vaccine composition provided herein, whereby the infectious disease in the subject is treated. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the administration is systemic administration or topical administration. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumoral administration, inhalation, subcutaneous administration, or intraperitoneal administration. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the administration is daily, weekly, monthly, or annually. In some embodiments, the administration is at least about every 6 hours, every 12 hours, every 24 hours (1 day), every 48 hours (2 days), every 72 hours (3 days), every 96 hours (4 days), every 120 hours (5 days), every 144 hours (6 days), every 168 hours (7 days), every 240 hours (10 days), every 336 hours (14 days), every 504 hours (21 days), every 672 hours (28 days), every 720 hours (30 days, 1 month), every 840 hours (35 days), every 1344 hours (56 days), every 8760 hours (365 days, 1 year), or at longer intervals. This specification further provides a method for the treatment of an infectious disease in a subject, the method further comprising administering to the subject an RNA vaccine composition. The infectious disease is a viral infectious disease. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the viral infectious disease is an upper respiratory viral infectious disease. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the infectious disease is a tumor viral infectious disease. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the upper respiratory viral infectious disease is COVID-19, SARS, MERS, influenza, parainfluenza, respiratory syncytial virus (RSV) infection, pneumonia, or rhinovirus infection. This specification further provides a method for the treatment of an infectious disease in a subject, wherein the infectious disease is a bacterial infectious disease, a fungal infectious disease, a parasitic infectious disease, or a yeast infectious disease.Also provided herein are methods 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 are methods for treating an infection in a subject, wherein the spike protein is derived from the SARS-CoV-2 virus. Also provided herein are methods for treating an infection in a subject, wherein the tumor viral infection is from human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (KSHV), Merkel cell polyomavirus (MCV), human T-cell lymphotropic virus type 1 (HTLV-1), or Epstein-Barr virus (EBV). Also provided herein are methods for treating an infection in a subject, wherein the subject is a mammal. Also provided herein are methods 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 the vaccine composition is administered to the subject.

[0136] The present specification provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the composition provided herein or the vaccine composition provided herein, whereby the cancer in the subject is treated. The present specification further provides a method for treating cancer, wherein the administration is systemic administration or local administration. The present specification further provides a method for treating cancer, wherein the administration is intramuscular administration, intravenous administration, oral administration, intradermal administration, intratumoral administration, inhalation, subcutaneous administration, or intraperitoneal administration. The present specification further provides a method for treating cancer, wherein the administration is daily, weekly, monthly, or yearly. In some embodiments, the administration is at least about every 6 hours, every 12 hours, every 24 hours (1 day), every 48 hours (2 days), every 72 hours (3 days), every 96 hours (4 days), every 120 hours (5 days), every 144 hours (6 days), every 168 hours (7 days), every 240 hours (10 days), every 336 hours (14 days), every 504 hours (21 days), every 672 hours (28 days), every 720 hours (30 days, 1 month), every 840 hours (35 days), every 1344 hours (56 days), or every 8760 hours (365 days, 1 year). The present specification further provides a method for treating cancer, the method further comprising administering to the subject an RNA vaccine composition. The present specification further provides a method for treating cancer, wherein the subject has cancer, is diagnosed with cancer, or is at risk of developing cancer. The present specification further provides a method for treating cancer, wherein the subject has a solid tumor or a hematological cancer. The present specification further provides a method for treating cancer, wherein the cancer is lung cancer, breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer, head and neck cancer, or thyroid cancer. The present specification further provides a method for treating cancer, wherein the composition comprises mRNA encoding a tumor antigen.Also provided herein are methods for the treatment of 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), preferentially expressed antigen in melanoma (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.

[0137] Preferred embodiments of the invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention.

Example

[0138] Example 1: Mechanisms of induction of innate and adaptive immune responses by RNA adjuvants. The functional mechanisms of RNA adjuvants are described below.

[0139] Figure 1 shows a schematic diagram of the approach of mRNA adjuvants for the improvement of mRNA vaccines. Figure 1 (upper panel) presents the mechanism of immune stimulation by a conventional mRNA vaccine, in which a lipid nanoparticle or carrier for RNA encoding an antigen is internalized by target cells and recognized by cellular signaling mechanisms such as TLR7 / 8 and RIG-1, thereby resulting in a downstream type I interferon response (IFN). In the case of a conventional mRNA vaccine, the mRNA is transported into host cells via lipid nanoparticles. The mRNA is translated into an antigen protein by the translation machinery of the host cell, which is then presented by the cell in conjunction with major histocompatibility complex (MHC) molecules. MHC class II molecules present the antigen to CD4+ helper T cells. MHC class I molecules present the antigen to CD8+ cytotoxic T cells. Subsequently, the interaction between the presented antigen and MHC class II molecules and the CD4+ helper T cell receptor activates CD4+ lymphocytes, releasing IL-2 and expressing IL-2 receptors on the surface of CD4+ lymphocytes. IL-2 produced by the activated cells stimulates not only the receptors of mononuclear phagocytes but also its own receptors, increasing their bactericidal activity. IL-2 also stimulates B cells to synthesize antibodies. B cells can recognize protein antigens in their native state, while T cells only recognize peptides resulting from antigen processing in the context of major histocompatibility complex molecules. Conventional vaccines may activate the production of interferon (IFN), which can interfere with efficient translation and reduce antigen production in host cells. IFN signaling triggers a Th-1 biased response.

[0140] Figure 1 (lower panel) shows mRNA that leads to NF-kB signaling via the stimulation of TLR5, TLR4, or TLR2, as well as the induction of the release of immunostimulatory cytokines and chemokines FlagThe mechanism of an adjuvant-driven mRNA vaccine is presented, which is expected to complement the mRNA-mediated IFN response and act in a synergistic manner to recruit an immune response to the antigen-producing site encoded by the mRNA vaccine. The balanced Th1-biased / Th2-biased immune response enabled by the TLR5 agonist adjuvant results in an improvement in the quality of the immune response.

[0141] Example 2: Generation, purification, and characterization of RNA adjuvants. The generation of RNA adjuvants and antigens encoded by RNA is described. All RNAs described are synthesized by in vitro transcription of DNA fragments encoding all elements. The cap structure can be covalently attached to the 5' UTR either co-transcriptionally or post-transcriptionally via different capping enzymes.

[0142] The final RNA product is purified using affinity chromatography with oligo dT to remove impurities (such as double-stranded RNA) generated during transcription that may strongly activate the innate immune response.

[0143] Additional modifications to RNA can be made. The untranslated regions (UTRs) of RNA are optimized using machine learning techniques trained on the ribosome loading profiles of a reporter gene library in which the 5’ UTR contains a completely random sequence. This is further tested and verified in a library of human UTRs and variants associated with diseases in humans. For 3’ UTR modification, a reporter gene can also be used and ligated to the 3’ end of random cDNAs obtained by reverse transcription of fragmented mRNAs isolated from human dendritic cells. By adding a second 3’ UTR from a different gene, the stability of the mRNA and protein expression can be further enhanced. In addition, RNA can be optimized by the poly-A tail by testing the levels of reporter proteins expressed from mRNAs with different poly-A tail lengths. Modified nucleobases can be used to further stabilize the RNA adjuvants and RNAs encoding antigens described herein.

[0144] The characterization of RNA can be performed using procedures selected from polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, and detection of RNA impurities, where the characterization includes determining the sequence of the RNA transcript, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript.

[0145] Example 3: RNA Synthesis and Purification. The mRNA compositions provided herein can be synthesized using T7 phage. Briefly, the mRNA is codon-optimized, synthesized, and cloned into an RNA production plasmid. After ligation into an expression vector, the mRNA is produced using T7 RNA polymerase in a linearized plasmid. Purification of the mRNA is performed via bead-based dialysis or spin column chromatography.

[0146] Example 4: RNA Complexation with Lipid Carriers. An RNA adjuvant and an mRNA encoding a SARS-CoV-2 spike protein are complexed with a lipid carrier. The lipid carrier can consist of an ionizable cationic lipid, cholesterol, a phospholipid (such as distearoyl phosphatidylcholine), and a polyethylene glycol (PEG)-lipid. The spike protein can include a signal sequence that targets the antigen to the endoplasmic reticulum of the cell, S1 and S2 domains, as well as 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 pre-fusion conformation of the spike protein.

[0147] The sequence of the RNA adjuvant is provided in Table 2 and the sequence section of this specification. The RNA can be admixed with the desired lipid carrier to form a vaccine composition. The resulting constructs of the RNA adjuvant and the RNA encoding the spike are provided in Figures 2A - 2D.

[0148] Construct 1: The RNA adjuvant is encapsulated within the same lipid carrier as the RNA encoding the antigen (Figure 2A).

[0149] Construct 2: The RNA adjuvant is encapsulated within a lipid carrier different from the RNA encoding the antigen (Figure 2B).

[0150] Construct 3: The RNA adjuvant and the RNA encoding the antigen are on the same nucleic acid strand and are encapsulated within a lipid carrier (Figure 2C).

[0151] Construct 4: The RNA is bicistronic. In this construct, the RNA sequence encoding the adjuvant is translated from an internal ribosome entry site (IRES), or the RNA adjuvant is separated from the antigen-encoding region of the RNA by a stop codon, and this construct allows for a reduction in the relative translation amount of the downstream open reading frame (Figure 2D).

[0152] Example 5: TLR5 agonist adjuvant composition and NF-κB signaling in vitro. mRNA encoding a TLR5 agonist adjuvant (TLR5 agonist 1) was generated by in vitro transcription (IVT) (SEQ ID NO: 2, SEQ ID NO: 176). mRNA was produced by transcribing a linearized plasmid DNA template (e.g., SEQ ID NO: 226). E. coli containing the plasmid was grown by fermentation in a bioreactor and then harvested to produce a purified plasmid. The mRNA was produced from the purified plasmid by T7 RNA polymerase as described above in Example 2 and Example 3.

[0153] HEK293 cells were infected with a lentivirus containing an NF-κB-driven luciferase reporter gene (hereinafter referred to as HEK293-NFkB-Luc cells) to generate a stable cell line. When the toll-like receptor (TLR) is activated by an agonist, luciferase activity increases in HEK293-NFkB-Luc cells as measured by a luminescence detector. Furthermore, when the TLR is activated by an mRNA adjuvant, the amount of secreted protein encoded by the mRNA adjuvant also increases as measured by enzyme-linked immunosorbent assay (ELISA).

[0154] To determine the effect of the mRNA adjuvant on TLR5-induced NF-κB signaling in vitro, HEK293-NFkB-Luc cells were transfected with TLR5 agonist 1 using polyethyleneimine (PEI) and compared to control HEK293-NFkB-Luc cells (null) (Figure 3A). Luciferase activity readings were performed and measured at 0, 18, and 40 hours after transfection with mRNA. NF-κB was activated by TLR5 agonist 1 compared to control cells (Figure 3B). Similarly, the protein secretion level of the TLR5 agonist increased in cells transfected with TLR5 agonist 1 compared to control cells at 18 and 40 hours after transfection (Figure 3C).

[0155] Next, to determine whether the packaging of lipid nanoparticles improves mRNA delivery, TLR5 agonist 1 mRNA was complexed with a lipid carrier. Lipid nanoparticles containing a cationic lipid surface, including PEG, DSPC, and cholesterol, were generated and purified.

[0156] 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 incubation (0 hours) and after 1-hour 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 hourly up to 49 hours after transfection (Figure 4A). Media were collected from each of the treated cell lines for ELISA analysis of TLR5 protein secretion. LNP without mRNA adjuvant (LNP(GFP)) did not activate the NF-κB-responsive luciferase reporter. In contrast, cell lines treated with either LNP-encapsulated mRNA TLR5 agonist (LNP(TLR5 agonist 1 RNA) or TLR5 agonist 1 protein showed increased luciferase activity compared to the control. This result demonstrated that the observed effect was specific to the TLR5 agonist adjuvant. These results also showed that LNP delivery of RNA encoding a TLR5 agonist elicited robust secretion of the TLR5 agonist protein compared to delivery of the protein alone or LNP control, towards enhancing TLR5 / NFκB cell signaling (Figure 4B).

[0157] 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 (Figures 5A - 5B). LNP (TLR5 agonist 1 RNA) increased both TLR5 luciferase activity (Figure 5A) and TLR5 agonist 1 protein secretion (Figure 5B) in human TLR5-expressing cells compared to cells not expressing human TLR5.

[0158] Example 6: In vivo distribution of LNP-mRNA adjuvant. Using a mouse model, the in vivo distribution of RNA delivered via lipid nanoparticles was tested. Twenty microliters of LNP-TLR5 agonist 1 (100 ng / μl of RNA), LNP-GFP, or PBS was injected intramuscularly (IM) into 38-week-old BALB / c-Tg(Rela-luc)31Xen females (Figure 6). After injection of the potassium salt of firefly D-luciferin (Gold Bio), animals were imaged for 5 minutes under anesthesia using a Perkin Elmer IVIS Spectrum in vivo imager. Total luminescence was measured at 0, 3, 5, 6, 8, 10, 12, 15, 18, and 36 hours post-injection for animals treated with LNP-TLR5 agonist 1, LNP-GFP, and PBS. Animals treated with LNP-TLR5 agonist mRNA showed high levels of firefly D-luciferin with a broad distribution of TLR5 protein throughout the animal (Figure 7). Production and secretion of the TLR5 agonist, as well as TLR5-dependent NF-kB activation, were confirmed in vitro and in vivo. No adverse events were detected in the treated animals.

[0159] It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within these claims and their equivalents be encompassed thereby.

Claims

1. A composition for inducing an immune response following administration to a target, wherein the composition is (a) an RNA adjuvant encoding a synthetic protein, wherein the synthetic protein comprises at least a functional fragment of a Toll-like receptor 5 (TLR5) agonist, (b) comprising one or more delivery vehicles formulated for intranasal, oral, intradermal, intranodal, intrasplenic, intratumoral, intravenous, or intramuscular administration, The composition, wherein an effective amount of the composition is administered to a subject in the presence of an antigen, followed by the induction of an initial innate immune response in the subject.

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

3. The composition according to claim 2, wherein the bacterial motile protein is a bacterial flagellin protein, a derivative thereof, or a functional fragment.

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

5. The composition according to 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 protein.

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

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

8. The composition according to claim 1, wherein the RNA comprises a sequence that is at least 85% identical to any one of sequence numbers 2 to 37.

9. The composition according to claim 1, wherein the one or more delivery vehicles are a diluent, an aqueous solution, particles, an emulsion, a suspension, a lipid carrier, a vesicle, a vector, or any combination thereof.

10. A vector comprising nucleic acids 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.

11. The vector according to claim 10, further comprising a nucleic acid sequence encoding a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor antigen.

12. A pharmaceutical composition comprising the composition according to any one of claims 1 to 9 and a carrier or excipient that is acceptable as a pharmaceutical.

13. A pharmaceutical composition comprising a vector according to any one of claims 10 to 11 and a carrier or excipient that is acceptable as a pharmaceutical.

14. A pharmaceutical composition for use as a pharmacopoeia to stimulate an immune response in a target, comprising the composition according to any one of claims 1 to 9 and a carrier or a pharmacopoeia-acceptable excipient, or the vector according to claim 10 or claim 11 and a carrier or a pharmacopoeia-acceptable excipient.

15. A pharmaceutical composition for use as a pharmacopoeia for the treatment of a disease or pathological condition in a subject, comprising the composition according to any one of claims 1 to 9 and a carrier or a pharmacopoeia-acceptable excipient, or the vector according to claim 10 or claim 11 and a carrier or a pharmacopoeia-acceptable excipient.

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