RNA composition for coding herpes simplex virus glycoprotein B antigen and application thereof

Abstract

The present disclosure provides a polyribonucleotide encoding a polypeptide comprising a viral antigen from herpes simplex virus-2 (HSV-2); for example, the viral antigens comprise an extracellular domain of HSV-2 glycoprotein B. The polyribonucleotides can be formulated as RNA compositions that can be used to induce an anti-HSV immune response in a subject.

Description

[0001] Sequence list declaration

[0002] This application contains a sequence list conforming to the rules of WIPO Standard ST.26, which has been electronically submitted in XML format and is hereby incorporated in its entirety by reference. The XML copy created on August 2, 2024, is titled P-682703-PC-SEQLIST2.xml and is 415 kilobytes in size.

[0003] Statement regarding federally sponsored research or development

[0004] This invention was carried out with the support of the U.S. government under license AI139618 granted by the National Institutes of Health. The government owns certain rights to this invention. Technical Field

[0005] This disclosure generally relates to the field of RNA therapeutics. In some embodiments, this disclosure provides an RNA construct encoding the HSV-2 glycoprotein as an immunotherapy for genital herpes. Background Technology

[0006] Globally, 500 million people are infected with herpes simplex virus type 2 (HSV-2). Many of these people are unaware of their infection but are at risk of transmitting it to their partners. Approximately 20% of those infected experience frequent, painful, recurrent genital lesions. Lifelong daily blocking therapy with acyclovir or valacyclovir can reduce the frequency of recurrences and lower the risk of transmission, but not everyone responds to or is willing to take daily medication. For people with genital herpes, the anxiety of transmitting the infection to their partners is likely the biggest concern.

[0007] One of the most frightening complications of genital herpes is neonatal herpes. This infection is uncommon (1 in 3,000 births in the United States) but devastating, with high morbidity and mortality rates in newborns. Newborns acquire herpes simplex virus type 1 (HSV-1) or HSV-2 infection from a mother whose infection is reactivated during delivery or from a pregnant woman who may have been infected for the first time in late pregnancy.

[0008] Preventative vaccines under development are designed to prevent first-time HSV infection, and those vaccines may not be effective in preventing relapse in those already infected. From a public health perspective, the greatest impact of an effective genital herpes vaccine will be on HIV infection. Genital herpes increases the risk of acquiring or transmitting HIV by 3-4 times. Summary of the Invention

[0009] In some embodiments, this document describes polynucleotides encoding polypeptides (e.g., nucleoside-modified polynucleotides) wherein the polypeptides comprise an extracellular domain of the herpes simplex virus-2 (HSV-2) glycoprotein B (gB) antigen or an immunogenic fragment thereof. In some embodiments, the extracellular domain of the HSV-2 gB comprises an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the polynucleotides disclosed herein comprise a ribonucleic acid sequence as shown in SEQ ID NO: 5.

[0010] In some embodiments, the polynucleotides disclosed herein further comprise an IL2 secretion signaling polypeptide; for example, a secretion signal sequence of an IL2 secretion signaling polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

[0011] In some embodiments, the polynucleotides disclosed herein further comprise a secretion signaling polypeptide encoding HSV gB; for example, a secretion signaling sequence of an HSV gB secretion signaling polypeptide having the amino acid sequence shown in SEQ ID NO: 4.

[0012] In some embodiments, the polynucleotides disclosed herein further include a 5' untranslated region. In some embodiments, the polynucleotides disclosed herein further include a 3' untranslated region. In some embodiments, the polynucleotides disclosed herein further include a polyA tail.

[0013] In some embodiments, the polynucleotides disclosed herein comprise a ribonucleic acid sequence as shown in SEQ ID NO: 6.

[0014] In another embodiment, this disclosure provides a composition or combination comprising the polynucleotides disclosed herein. In some embodiments, the composition or combination comprises nanoparticles, lipids, polymers, cholesterol, or cell-penetrating peptides encapsulating the polynucleotides.

[0015] In some embodiments, the composition or combination further comprises one or more RNAs encoding the following:

[0016] a) HSV glycoprotein C (gC) or its immunogenic fragment,

[0017] b) HSV glycoprotein D (gD) or its immunogenic fragment,

[0018] c) HSV glycoprotein E (gE) or its immunogenic fragment,

[0019] d) HSV glycoprotein H (gH) or its immunogenic fragment,

[0020] e) HSV glycoprotein L (gL) or its immunogenic fragment,

[0021] f) HSV glycoprotein I (gI) or its immunogenic fragment, or

[0022] g) Any combination thereof.

[0023] In another embodiment, a method is provided for treating a subject with herpes simplex virus (HSV) infection using the polynucleotides disclosed herein or the compositions or combinations disclosed herein.

[0024] In another embodiment, a method is provided for using the polynucleotides disclosed herein or the compositions or combinations disclosed herein to inhibit or suppress herpes simplex virus (HSV) infection in a subject or reduce its incidence.

[0025] In another embodiment, a method is provided for inducing an anti-HSV immune response in a subject using the polynucleotides disclosed herein or the compositions or combinations disclosed herein.

[0026] These and other aspects of the invention will be understood from the following description of the drawings and the detailed description of the invention. Attached Figure Description

[0027] This document describes some embodiments of the invention by way of example only, with reference to the accompanying drawings. Specific details are now shown in particular with regard to the drawings, with emphasis placed on the details shown by way of example and for the purpose of illustrative discussion of embodiments of the invention. In this respect, the description taken in conjunction with the drawings will make it clear to those skilled in the art how to practice embodiments of the invention.

[0028] Figure 1 The cumulative number of days of recurrent genital lesions is shown for each group. Previously infected guinea pigs were immunized on days 35 and 65 post-infection with either an exemplary immunogenic fragment (30 μg) encapsulated in lipid nanoparticles 315 and expressing HSV-2 gB (gB2) or nucleoside-modified RNA in PBS (control). Recurrent genital lesions were scored daily, Monday through Friday, from day 1 post-immunization until the end of the study on day 116. Significant differences were observed between the gB2 group and the PBS group starting from the second immunization.

[0029] Figure 2 The number of days from one day after the second immunization for recurrent genital lesions is shown for each animal. The animals shown here are compared to... Figure 1 The results were identical to those in the PBS group. P-values ​​were calculated using the two-tailed Mann-Whitney test and showed a highly significant difference between the gB2 group and PBS (**, P < 0.01).

[0030] Figure 3 This study demonstrates cytokine production by CD4+ T cells in response to a composition containing HSV-2 glycoprotein. Mice were immunized twice with an exemplary immunogenic fragment of HSV-2 gB (gB2) RNA-LNP, 10 μg, as described herein. Spleen cells from these mice were stimulated with a gB2 overlapping peptide library. T cells producing CD4+ cytokines were analyzed by flow cytometry.

[0031] Figure 4 This study demonstrates cytokine production by CD8+ T cells in response to a composition containing HSV-2 glycoprotein. Mice were immunized twice with an exemplary immunogenic fragment of HSV-2 gB (gB2) RNA-LNP, 10 μg, as described herein. Spleen cells from these mice were stimulated with a gB2 overlapping peptide library. T cells producing CD8+ cytokines were analyzed by flow cytometry.

[0032] Figure 5 The expression levels in HEK293T cells transfected with RNA encoding the HSV-2 gB (gB2) antigen are shown. Constructs encoding the gB2 antigen include 4059 (SEQ ID NO: 219), 4060 (SEQ ID NO: 220), 4061 (SEQ ID NO: 221), 4063 (SEQ ID NO: 225), 4064 (SEQ ID NO: 226), and 4065 (SEQ ID NO: 227). Representative data from one experiment show the median fluorescence intensity (MFI) of the total HEK293T population used for the gB2 antigen constructs. The data shown are the mean + standard deviation of HEK293T transfections performed in triplicate.

[0033] Figures 6A-6D The survival rates of BNT163, gB, gE2 / gI2, or combinations thereof in HSV-2-infected guinea pigs are shown. Figure 6A ), severity of disease ( Figure 6B ), genital lesions ( Figure 6C ) and urinary retention ( Figure 6D The preventive effect of ( ). For example Figures 6A-6D As shown, “BNT163” refers to a combination of three polynucleotides: (i) a polynucleotide encoding a polypeptide containing a gC immunogenic fragment having an amino acid sequence according to SEQ ID NO: 258; (ii) a polynucleotide encoding a polypeptide containing a gD immunogenic fragment having an amino acid sequence according to SEQ ID NO: 260; and (iii) a polynucleotide encoding a polypeptide containing a gE immunogenic fragment having an amino acid sequence according to SEQ ID NO: 262.

[0034] Figures 7A-7B The therapeutic effects of BNT163, gB, and gE2 / gI2 on recurrent lesions in the initial trial were demonstrated. Figure 7A ) and the combined results of the two experiments ( Figure 7B ).like Figures 7A-7B The term “BNT163” as used refers to a combination of three polynucleotides: (i) a polynucleotide encoding a polypeptide containing a gC immunogenic fragment having an amino acid sequence according to SEQ ID NO: 258; (ii) a polynucleotide encoding a polypeptide containing a gD immunogenic fragment having an amino acid sequence according to SEQ ID NO: 260; and (iii) a polynucleotide encoding a polypeptide containing a gE immunogenic fragment having an amino acid sequence according to SEQ ID NO: 262.

[0035] Figures 8A-8B It shows that in CD4+ ( Figure 8A ) and CD8+ Figure 8B T cell responses to immunization with gB2 RNA. Detailed Implementation

[0036] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. While similar or equivalent materials and methods to those described and used herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including its definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Every reference or other citation mentioned herein is incorporated herein by reference in its entirety.

[0037] In the description provided herein, every step of the invention and its variations have been described. This description is not intended to be limiting, and changes to components, the order of steps, and other variations should be understood to fall within the scope of the invention.

[0038] It should be understood that, for clarity, certain features of the invention described in the context of a single embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination or as appropriate in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments are invalid without those elements.

[0039] Definitions

[0040] As used herein, unless the context clearly indicates otherwise, the singular forms “a”, “an” and “the” include plural indicators.

[0041] As used herein, the term "about" when used with reference values ​​refers to a value similar to the reference value in the context. Generally, those skilled in the art will understand the extent of variation covered by "about" in said context. For example, in some embodiments, the term "about" may cover values ​​within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0042] As used herein, the term "adjuvant" refers to a compound that, when administered to an individual or tested in vitro, enhances the immune response to an antigen in an individual or test system to which the antigen is administered. In some embodiments, an immune adjuvant enhances the immune response to an antigen that is weakly immunogenic when administered alone, i.e., that induces no or low antibody titers or a cell-mediated immune response. In some embodiments, an adjuvant increases the antibody titer against the antigen. In some embodiments, an adjuvant reduces the dose of the antigen required to achieve an effective immune response in an individual. Many types of adjuvants are known in the art and are described in detail in U.S. Patent Publication 2013 / 0028925, which is hereby incorporated by reference.

[0043] As used herein, the term "administration" means the direct introduction of the composition of this disclosure into a subject by injection or other means. In another embodiment, "administration" means contacting cells of a subject's immune system with the composition encoding HSV gE and gI or a modified polynucleotide.

[0044] As used herein, the term "pharmaceutical" can refer to a physical entity or phenomenon. In some embodiments, a pharmaceutical can be characterized by specific features and / or effects. In some embodiments, a pharmaceutical can be a compound, molecule, or entity of any chemical class, including, for example, small molecules, peptides, nucleic acids, carbohydrates, lipids, metals, or combinations or complexes thereof. In some embodiments, the term "pharmaceutical" can refer to a compound, molecule, or entity comprising a polymer. In some embodiments, the term can refer to a compound or entity comprising one or more polymeric moieties. In some embodiments, the term "pharmaceutical" can refer to a compound, molecule, or entity substantially free of a particular polymer or polymeric moieties. In some embodiments, the term can refer to a compound, molecule, or entity lacking or substantially free of any polymer or polymeric moieties.

[0045] As used herein, the term "amino acid" in its broadest sense refers to a compound and / or substance that may be, is, or has been incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, an amino acid has the universal structure H₂N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including the carboxyl group and / or the amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the universal structure described above. For example, in some embodiments, an amino acid may be modified compared to the universal structure by methylation, amidation, acetylation, PEGylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, carboxyl group, one or more protons, and / or hydroxyl groups). In some embodiments, such modification can, for example, alter the cyclic half-life of a peptide containing modified amino acids compared to a peptide containing otherwise identical unmodified amino acids. In some embodiments, such modification does not significantly alter the relevant activity of a peptide containing modified amino acids compared to a peptide containing otherwise identical unmodified amino acids. As will be apparent from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in other embodiments, the term may be used to refer to an amino acid residue of a peptide.

[0046] As used herein, the term "antibody agent" generally refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes an immunoglobulin structural element sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) substantially identical to a CDR found in a reference antibody. In some embodiments, the included CDR is substantially identical to a reference CDR because it has the same sequence or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to a reference CDR because it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR because 1-5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR because at least one amino acid in the included CDR is substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the included CDR is substantially identical to the reference CDR because 1-5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR in other respects. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements that are recognized by those skilled in the art as variable domains of immunoglobulins.In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as corresponding to CDR1, 2, and 3 of the antibody variable domain; in some such embodiments, the antibody agent is or comprises a polypeptide or a group of polypeptides whose amino acid sequence together includes structural elements recognized by those skilled in the art as corresponding to the heavy chain and light chain variable region CDRs, for example, heavy chain CDR1, 2, and / or 3 and light chain CDR1, 2, and / or 3. In some embodiments, the antibody agent is a polypeptide protein having a binding domain homologous or substantially homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent may be or comprises a polyclonal antibody preparation. In some embodiments, the antibody agent may be or comprises a monoclonal antibody preparation. In some embodiments, the antibody agent may include one or more constant region sequences specific to a particular organism (such as a camel, human, mouse, primate, rabbit, or rat); in many embodiments, the antibody agent may include one or more constant region sequences specific to humans. In some embodiments, the antibody agent may include one or more sequence elements that would be recognized by those skilled in the art as humanized sequences, primate-like sequences, chimeric sequences, etc. In some embodiments, the antibody agent may be a typical antibody (e.g., it may contain two heavy chains and two light chains). In some embodiments, the antibody agent may be in a format selected from, but not limited to, the following: complete IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masking antibodies (e.g., Probodies®). Small type Mod Block Immune medicine Material (SMIPs) TMThe following are examples of antibodies: single-chain or tandem bivalent antibodies (TandAb®); VHH; Anticalins®; Nanobodies® microantibodies; BiTE®; ankylosing spicules or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack the covalent modifications (e.g., glycosyl attachment) that are naturally present. In some embodiments, the antibody may contain covalent modifications (e.g., glycosyl attachment), payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.) or other side groups (e.g., polyethylene glycol, etc.).

[0047] As used herein, the term "antigen" refers to a molecule recognized by the immune system, for example, in a specific embodiment, the adaptive immune system, such that the antigen induces an antigen-specific immune response. In some embodiments, an antigen-specific immune response may be or includes antibody-producing and / or antigen-specific T cells. In some embodiments, an antigen is a peptide or polypeptide containing at least one epitope against which an immune response can be generated. In some embodiments, the antigen is presented by cells of the immune system, such as antigen-presenting cells like dendritic cells or macrophages. In one embodiment, the antigen or a processed product thereof (such as a T-cell epitope) is bound to T-cell or B-cell receptors, or to immunoglobulin molecules (such as antibodies). Thus, the antigen or a processed product thereof may specifically react with antibodies or T lymphocytes (T cells). In some embodiments, the antigen is a parasitic antigen. According to this disclosure, in some embodiments, the antigen may be delivered by RNA molecules as described herein. In some embodiments, the length of the peptide or polypeptide antigen can be 2-100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the peptide or polypeptide antigen can be greater than 50 amino acids. In some embodiments, the peptide or polypeptide antigen can be greater than 100 amino acids. In some embodiments, the antigen is recognized by immune effector cells. In some embodiments, the antigen recognized by immune effector cells is capable of inducing stimulation, activation, and / or amplification of immune effector cells carrying antigen receptors that recognize the antigen in the presence of appropriate co-stimulatory signals. In the context of embodiments of this disclosure, in some embodiments, the antigen can be presented or present on the surface of cells, such as antigen-presenting cells. In some embodiments, the antigen is presented by diseased cells, such as virus-infected cells. In some embodiments, the antigen receptor is a TCR that binds to the epitope of the antigen presented in the MHC context. In some embodiments, when a TCR is expressed by and / or present on T cells, its binding to an antigen presented by a cell (such as an antigen-presenting cell) causes stimulation, activation, and / or expansion of the T cells. In some embodiments, when a TCR is expressed by and / or present on T cells, its binding to an antigen presented on a lesion cell causes cytolysis and / or apoptosis of the lesion cell, wherein the T cell preferably releases cytotoxic factors, such as perforin and granzyme.

[0048] As used herein, the term “antigenic” refers to a peptide that is capable of specifically interacting with antigen recognition molecules of the immune system, such as immunoglobulins (antibodies) or T-cell antigen receptors.

[0049] As used herein, two events or entities are “related” to each other if the presence, level, extent, type, and / or form of one event or entity is related to the presence, level, extent, type, and / or form of another event or entity, as the term is used herein. For example, if the presence, level, and / or form of a particular entity (e.g., peptide, genetic signature, metabolite, microorganism, etc.) is related to the incidence, susceptibility, severity, stage, etc., of a disease, condition, or symptom (e.g., across relevant populations), the particular entity is considered related to the particular disease, condition, or symptom. In some embodiments, two or more entities are physically “associated” to each other if they interact directly or indirectly such that they are physically close to each other and / or remain close to each other. In some embodiments, two or more physically associated entities are covalently linked to each other; in some embodiments, two or more physically related entities are not covalently linked to each other, but are non-covalently associated, for example, through hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0050] As used herein, the term "binding" generally refers to non-covalent association between or within entities or parts. In some embodiments, binding data are expressed as "IC50". As understood in the art, IC50 is the concentration of the agent evaluated in a binding assay at which a 50% binding inhibition of a reference agent known to bind to the relevant binding pair is observed at the stated concentration. In some embodiments, the assay is run under specific conditions (e.g., limiting the binding target and reference concentrations) such that these values ​​are close to K. D Values. Assays used to determine binding are well known in the art and are described in detail in, for example, the following publications: PCT Publications WO 94 / 20127 and WO 94 / 03205, and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney et al., Journal of Immunology 154:247 (1995); and Sette et al., Molecular Immunology 31:813 (1994). Alternatively, binding can be expressed relative to the binding of a reference standard peptide. For example, it can be based on its IC50 value. 50 IC50 relative to reference standard peptide 50The binding can also be determined using other assay systems, including those using live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., International Journal of Immunology 2:443 (1990); Hill et al., Journal of Immunology 147:189 (1991); del Guercio et al., Journal of Immunology 154:685 (1995)); cell-free systems using detergent lysis buffers (e.g., Cerundolo et al., Journal of Immunology 21:2069 (1991)); and immobilized purified MHC (e.g., Hill et al., Journal of Immunology 152, 2890 (1994); Marshall et al., Journal of Immunology 152:4946). (1994)), ELISA systems (e.g., Reay et al., Journal of the European Molecular Biology Society (EMBO J) 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., Journal of Biol. Chem. 268:15425 (1993)); high-throughput soluble phase assays (Hammer et al., Journal of Experimental Medicine 180:2353 (1994)); and measurements of class I MHC stability or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., Journal of Immunology 149:1896 (1992)).

[0051] As used herein, the term "C5 interference domain" refers to a domain that interferes with the binding of host C3b molecules to host C5 molecules. In another embodiment, the term refers to a domain that interferes with the interaction between host C3b molecules and host C5 molecules.

[0052] As used herein, the term "cap" refers to a structure comprising or substantially composed of nucleoside-5'-triphosphate, which is typically attached to the 5' end of capless RNA (e.g., capless RNA with a 5'-diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, for example, including but not limited to a 7-methylguanosine cap, structurally named "m7G". In some embodiments, the cap is or comprises a synthetic cap analogue with a structure similar to that of the RNA cap and has the ability to stabilize it when attached to RNA, for example, including but not limited to anti-reverse cap analogues (ARCA) known in the art. Those skilled in the art will understand that methods for attaching a cap to the 5' end of RNA are known in the art. For example, in some embodiments, capped RNA can be obtained by in vitro capping of RNA with a 5' triphosphate group or RNA with a 5' diphosphate group using a capping enzyme system (e.g., including but not limited to a vaccinia capping enzyme system or a Saccharomyces cerevisiae capping enzyme system). Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a single-stranded DNA template in the presence of a dinucleotide or trinucleotide cap analogue.

[0053] As used herein, the term “co-administration” refers to the use of a pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) in conjunction with an additional therapeutic agent. The combined use of a pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) with an additional therapeutic agent may be performed simultaneously or at intervals (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) and an additional therapeutic agent may be combined in a pharmaceutically acceptable carrier, or may be placed in a separate carrier and delivered to target cells or administered to a subject at different times. Each of these cases is considered to fall under the meaning of “co-administration” or “combination”, provided that the pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) and the additional therapeutic agent are delivered or administered sufficiently close in time to result in at least a temporal overlap in the biological effects on the target cells or the treated subject.

[0054] As used herein, the term "codon optimization" refers to altering codons in the coding region of a nucleic acid molecule to reflect typical codon usage in the host organism. In some embodiments, codon optimization does not alter the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, in some embodiments, the coding region is codon-optimized using the RNA molecule described herein for optimal expression in a subject to be treated. In some embodiments, codon optimization may be performed such that a codon that yields high-frequency tRNA is inserted in place of a "rare codon". In some embodiments, codon optimization may include increasing the G / C content of the coding region of the RNA described herein, relative to the guanosine / cytosine (G / C) content of the corresponding coding sequence of wild-type RNA, wherein the amino acid sequence encoded by the RNA is unmodified compared to the amino acid sequence.

[0055] As used herein, the term "combination" refers to two or more pharmaceutical agents (e.g., two or more polynucleotides) or compositions (e.g., a therapeutic composition) used together. In some embodiments, two or more pharmaceutical agents (e.g., two or more polynucleotides) or compositions (e.g., a therapeutic composition) are used together (e.g., prepared, formulated, marketed, or administered). For example, in some embodiments, two or more pharmaceutical agents or compositions may be used as part of the same treatment or prevention regimen (e.g., prepared, formulated, marketed, or administered). In some embodiments, two or more pharmaceutical agents or compositions may be administered to a subject simultaneously or nearly simultaneously (e.g., within 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, 5 minutes or less, or 1 minute or less). For clarity, it is not necessary to use a single agent or composition together in a single composition (e.g., to prepare, formulate, market, or apply); however, in some embodiments, two or more agents or compositions may be used together in a single composition (e.g., to prepare, formulate, market, or apply).

[0056] As used herein, the term "combination therapy" refers to those situations where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "dose" of the first regimen are administered before any dose of the second regimen is administered); in some embodiments, such regimens are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may involve administering one or more agents or forms to a subject receiving other agents or forms in the combination. For clarity, combination therapy does not require the individual agents to be administered together in a single composition (or even necessarily simultaneously), but in some embodiments, two or more agents or their active portions may be administered together in the combination composition.

[0057] As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be exactly identical to each other but are similar enough to allow comparisons between them such that a person skilled in the art will understand that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, comparable sets of conditions, situations, individuals, or groups are characterized by a number of substantially identical features and one or a few different features. A person skilled in the art will understand, in context, the degree of identity required for two or more such agents, entities, situations, sets of conditions, etc., to be considered comparable in any given situation. For example, a person skilled in the art will understand that a set of situations, individuals, or groups is comparable to each other when it is characterized by a sufficient number and type of substantially identical features to ensure that the following reasonable conclusion is reached: the difference in results or observed phenomena obtained under different sets of situations, individuals, or groups, or using different sets of situations, individuals, or groups, is caused by or indicates a change in those features that have changed.

[0058] As used herein, the terms “comprise,” “comprises,” “comprising,” “includes,” “including,” “having,” and their cognates mean “including but not limited to.”

[0059] As used herein, the term "composed of" means "including and limited to". The term "generally composed of" means that a composition, method, or structure may include additional ingredients, steps, and / or portions, provided that the additional ingredients, steps, and / or portions do not substantially alter the essential and novel characteristics of the claimed composition, method, or structure.

[0060] As used herein, the term "generally composed of" means that a composition, method, or structure may include additional components, steps, and / or portions, provided that the additional components, steps, and / or portions do not substantially alter the essential and novel characteristics of the claimed composition, method, or structure.

[0061] As used herein, the term "corresponds to" refers to a relationship between two or more entities. For example, the term "corresponds to" can be used to indicate the positional / identical nature of a structural element in a compound or composition relative to another compound or composition (e.g., relative to a suitable reference compound or composition). For example, in some embodiments, monomeric residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as "corresponding to residues in a suitable reference polymer." For example, those skilled in the art will understand that, for simplicity, residues in a polypeptide are typically named using a typical numbering system based on a reference-related polypeptide, so the amino acid "corresponding to" a residue at position 190 does not necessarily have to be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue found at position 190 in the reference polypeptide; those skilled in the art readily understand how to identify "corresponding" amino acids. For example, those skilled in the art will understand various sequence alignment strategies, including software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which can be used, for example, to identify “corresponding” residues in peptides and / or nucleic acids according to this disclosure. Those skilled in the art will also understand that, in some cases, the term “corresponding” can be used to describe an event or entity that has a related similarity to another event or entity (e.g., a suitable reference event or entity). To cite just one example, a gene or protein in one organism may be described as “corresponding” to a gene or protein from another organism, indicating that in some embodiments, the gene or protein plays a similar role or performs a similar function and / or exhibits a particular degree of sequence identity or homology, or shares specific characteristic sequence elements.

[0062] As used herein, in the context of an amino acid sequence (peptide or polypeptide), “derived from” a specified amino acid sequence (peptide or polypeptide) means a structural analog of the specified amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has the same, substantially the same, or homologous amino acid sequence as said particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of said particular sequence or a fragment thereof. For example, those skilled in the art will understand that antigens applicable herein may be modified such that they differ in sequence from their naturally occurring or native sequence from which they originate, while retaining the desired activity of the native sequence.

[0063] As used herein, the term “designed” refers to a pharmaceutical agent that: (i) has a structure that is artificially selected or has been artificially selected; (ii) is produced by a process that requires artificial intervention; and / or (iii) is different from natural substances and other known pharmaceutical agents.

[0064] As used herein, the term "dosing regimen" can be used to refer to a set of unit doses (typically more than one unit dose) administered individually to a subject at regular intervals. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, a dosing regimen comprises multiple doses, each of which is time-interval with the other doses. In some embodiments, individual doses are spaced apart from each other by the same length of time; in some embodiments, a dosing regimen comprises multiple doses and at least two different time intervals between individual doses. In some embodiments, all doses within a dosing regimen have the same unit dose. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen comprises a first dose of a first dose, followed by one or more additional doses of a second dose different from the first dose. In some embodiments, a dosing regimen comprises a first dose of a first dose, followed by one or more additional doses of a second dose identical to the first dose. In some embodiments, when administered in a relevant population, the dosing regimen is associated with a desired or beneficial outcome (i.e., it is a therapeutic dosing regimen).

[0065] As used herein, “encoding” refers to an RNA molecule containing a gene or a segment thereof encoding the protein of interest. In another embodiment, the RNA molecule comprises or is composed of a protein-coding sequence or a segment thereof encoding the protein of interest. In another embodiment, it also encodes one or more other proteins or segments thereof. In yet another embodiment, the protein of interest or a segment thereof uniquely encodes the protein. Each possibility represents a separate embodiment of this disclosure.

[0066] As used herein, the term “engineered” refers to aspects that have been artificially manipulated. For example, a polynucleotide is considered “engineered” when two or more sequences that are not naturally linked together in the specified order are artificially manipulated to directly link to each other in an engineered polynucleotide and / or when a particular residue in a polynucleotide is not naturally present and / or is artificially linked to an entity or portion that is not naturally linked to it.

[0067] As used herein, the term "epitope" refers to a portion that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope can be recognized by T cells, B cells, or antibodies. In some embodiments, an epitope comprises a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on a surface when the antigen takes an associated three-dimensional configuration. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen takes such a configuration. In some embodiments, at least some of such chemical atoms are groups that are physically separated from each other when the antigen takes an alternative configuration (e.g., linearization). Thus, in some embodiments, an epitope of an antigen may comprise continuous or discontinuous portions of the antigen. In some embodiments, an epitope is or comprises a T-cell epitope. In some embodiments, the length of an epitope may be about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0068] As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from said nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence involves one or more of the following: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translating RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0069] As used herein, the term “5' UTR” refers to the sequence of an RNA molecule between the transcription start site and the start codon of the coding region. In some embodiments, “5' UTR” refers to the sequence of an RNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (typically AUG) of the coding region of the RNA molecule, for example, in its natural background.

[0070] As used herein, the term "fragment" refers to a protein or polypeptide that is shorter than a full-length protein or polypeptide or contains fewer amino acids. In another embodiment, a fragment refers to a nucleic acid that is shorter than a full-length nucleic acid or contains fewer nucleotides encoding a protein fragment. In one embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment of an HSV protein is an extracellular domain of the protein. In another embodiment, the fragment is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids shorter than the full-length protein. In another embodiment, the fragment is 50-100, 100-150, 150-300, or 300-600 amino acids shorter than the full-length protein. In another embodiment, the fragment comprises approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the full-length protein. In another embodiment, the fragment comprises approximately 42%, 83%, 78%, or 66% of the full-length protein (excluding the signal sequence), as described herein, and in one embodiment, for HSV-2 gE, gC, gD, and gI.

[0071] In some embodiments, the fragment is a segment of a protein, peptide, or nucleic acid. In another embodiment, the fragment is an immunogenic segment of a protein, peptide, or nucleic acid. In another embodiment, the fragment is a functional segment within a protein, peptide, or nucleic acid. In another embodiment, the fragment is an N-terminal immunogenic fragment. In some embodiments, the fragment is a C-terminal immunogenic fragment. In another embodiment, the fragment is an N-terminal functional fragment. In another embodiment, the fragment is a C-terminal functional fragment. In another embodiment, the fragment comprises fragments of proteins linked together or fragments of multiple proteins linked together. In some embodiments, the fragment is a domain (e.g., an extracellular domain).

[0072] As used herein, the term "functional" refers to the innate ability of a protein, peptide, nucleic acid, fragment thereof, or variant thereof to exhibit biological activity or function. In some embodiments, such biological function is its binding property with an interacting partner (e.g., a membrane-associated receptor), and in another embodiment, its trimerization property. In the cases of functional fragments and functional variants of this disclosure, these biological functions can indeed be altered (e.g., regarding their specificity or selectivity) while retaining the essential biological function.

[0073] As used herein, in one embodiment, the terms “homology,” “homology,” etc., when relating to any protein or peptide, refer to the percentage of amino acid residues in a candidate sequence that are identical to residues of the corresponding native polypeptide, after sequence alignment and cleavage (if necessary) to achieve the maximum percentage of homology, and without considering any conserved substitutions as part of sequence identity. Methods and computer programs used for alignment are well known in the art.

[0074] In some embodiments, "homology" means that the protein sequence encoded by a modified (e.g., nucleoside-modified) polynucleotide shares greater than 70% identity with the sequence disclosed herein. In another embodiment, the identity is greater than 72%. In another embodiment, the identity is greater than 75%. In another embodiment, the identity is greater than 78%. In another embodiment, the identity is greater than 80%. In another embodiment, the identity is greater than 82%. In another embodiment, the identity is greater than 83%. In another embodiment, the identity is greater than 85%. In another embodiment, the identity is greater than 87%. In another embodiment, the identity is greater than 88%. In another embodiment, the identity is greater than 90%. In another embodiment, the identity is greater than 92%. In another embodiment, the identity is greater than 93%. In another embodiment, the identity is greater than 95%. In another embodiment, the identity is greater than 96%. In another embodiment, the identity is greater than 97%. In another embodiment, the identity is greater than 98%. In another embodiment, the identity is greater than 99%. In another embodiment, the identity is 100%.

[0075] As used herein, the term "HSV-1" refers to herpes simplex virus-1. In some embodiments, "HSV-1" refers to a specific HSV-1 strain. In another embodiment, the term refers to the KOS strain. In another embodiment, the term refers to the F strain. In another embodiment, the term refers to the NS strain. In another embodiment, the term refers to the CL101 strain. In another embodiment, the term refers to the "17" strain. In another embodiment, the term refers to the "17+syn" strain. In another embodiment, the term refers to the MacIntyre strain. In another embodiment, the term refers to the MP strain. In another embodiment, the term refers to the HF strain. In another embodiment, the term refers to any other HSV-1 strain known in the art.

[0076] As used herein, the term "HSV-2" refers to herpes simplex virus-2. In some embodiments, "HSV-2" refers to a specific HSV-2 strain. In another embodiment, the term refers to HSV-2 strain 333. In another embodiment, the term refers to strain 2.12. In another embodiment, the term refers to strain HG52. In another embodiment, the term refers to strain MS. In another embodiment, the term refers to strain G. In another embodiment, the term refers to strain 186. In yet another embodiment, the term refers to any other HSV-2 strain known in the art.

[0077] As used herein, the term "humoral immunity" or "humoral immune response" refers to antibody production and its associated auxiliary processes, including: Th2 activation and cytokine production, germinal center formation and allotype conversion, affinity maturation, and memory cell generation. Humoral immunity also refers to the effector functions of antibodies, including pathogen neutralization, classical complement activation, and opsonins that promote phagocytosis and pathogen clearance.

[0078] As used herein, the term "identity" refers to the overall association between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, molecules are considered "substantially identical" to each other if the sequences of polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The percentage of identity between two sequences can be calculated, for example, by aligning two nucleic acid or polypeptide sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second sequences, and dissimilar sequences can be discarded for comparison purposes). In some embodiments, the length of the sequences compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, 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 substantially 100% of the length of the reference sequence. Nucleotides at corresponding positions are then compared. The molecules are considered identical at said position when a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies and the length of each vacancy that needs to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. For example, the algorithm of Meyers and Miller, 1989, which has been integrated into the ALIGN program (version 2.0), can be used to determine the percentage of identity between two nucleotide sequences. In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program employ a PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the GAP program in the GCG software package can be used with the NWSgapdna.CMP matrix to determine the percentage of identity between two nucleotide sequences.

[0079] As used herein, the term "immune escape domain" refers to a domain that interferes with or reduces the in vivo anti-HSV efficacy of an anti-HSV antibody (e.g., an anti-gE antibody). In some embodiments, the domain interferes with or reduces the in vivo anti-HSV efficacy of an anti-HSV immune response. In some embodiments, the domain reduces the immunogenicity of an HSV protein (e.g., gE) during subsequent infection. In some embodiments, the domain reduces the immunogenicity of an HSV protein during subsequent challenge. In some embodiments, the domain reduces the immunogenicity of HSV during subsequent challenge. In some embodiments, the domain reduces the immunogenicity of an HSV protein in the context of persistent HSV infection. In some embodiments, the domain reduces the immunogenicity of HSV in the context of persistent HSV infection. In some embodiments, the domain functions as an IgG Fc receptor. In some embodiments, the domain promotes antibody bipolar bridging, which in one embodiment refers to the ability of an antibody molecule to bind to an HSV antigen via its Fab domain and to a separate HSV antigen, such as gE in one embodiment, via its Fc domain, thereby preventing the Fc domain from activating complement.

[0080] As used herein, the term "immunogenic fragment" refers to a portion of a protein, which in one embodiment is immunogenic and in other embodiments induces a protective immune response when administered to a subject.

[0081] As used herein, the term "immunogenicity" or "immunogenicity" refers to the inherent ability of a protein, peptide, nucleic acid, antigen, or organism to induce an immune response in an animal when administered to the animal. In one embodiment, an immunogenic peptide also has antigenicity. Therefore, in some embodiments, "enhanced immunogenicity" refers to an increased ability of a protein, peptide, protein fragment, nucleic acid, antigen, or organism to induce an immune response in an animal when administered to the animal. In some embodiments, the enhanced ability of a protein, peptide, protein fragment, nucleic acid, antigen, or organism to induce an immune response can be measured by: a larger number of antibodies against the protein, peptide, protein fragment, nucleic acid, antigen, or organism; a greater diversity of antibodies against the antigen or organism; a larger number of T cells specific to the protein, peptide, protein fragment, nucleic acid, antigen, or organism; greater cytotoxicity or helper T cell responses against the protein, peptide, nucleic acid, antigen, or organism, etc.

[0082] As used herein, the term "immunologic equivalence" means that immunologically equivalent molecules (such as immunologically equivalent amino acid sequences) exhibit the same or substantially the same immunological properties and / or exert the same or substantially the same immunological effects, for example, in terms of the type of immunological effect. In the context of this disclosure, in some embodiments, the term "immunologic equivalence" is used to describe the immunological effects or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, upon exposure to the immune system of a subject, it induces an immune response that is reaction-specific to a reference amino acid sequence.

[0083] In some embodiments, the antigen receptor is an antibody or B-cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B-cell receptor binds to the natural epitope of the antigen.

[0084] As used herein, these terms, or grammatically equivalent comparative terms, refer to values ​​relative to equivalent reference measurements. For example, in some embodiments, the value of an assessment obtained with the provided pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) may be “increased” relative to a value obtained with an equivalent reference pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine). Alternatively or additionally, in some embodiments, the assessment value obtained in a subject may be “increased” compared to results obtained in the same subject under different conditions (e.g., before or after an event; or with or without an event, such as administration of a pharmaceutical composition as described herein (e.g., an immunogenic composition, such as a vaccine)); or in different equivalent subjects (e.g., in equivalent subjects who differ from the subject of interest in their prior exposure to conditions, such as not having been administered a pharmaceutical composition as described herein (e.g., an immunogenic composition, such as a vaccine)). In some embodiments, equivalent terms refer to statistically relevant differences (e.g., the incidence of the difference and / or the magnitude sufficient to achieve statistical significance). Those skilled in the art will recognize, or will be able to readily determine, in a given context, the degree and / or incidence of difference required or sufficient to achieve such statistical significance. In some embodiments, the term "reduction" or equivalent means that the level of the assessed value is reduced by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or more compared to a comparable reference. In some embodiments, the term "reduction" or equivalent means complete or substantially complete suppression, i.e., reduction to zero or substantially zero. In some embodiments, the term "increase" or "induction" means that the level of the assessed value is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or more compared to a comparable reference.

[0085] As used herein, the term "ionizable" refers to a compound, group, or atom that is charged at a certain pH. In the context of ionizable amino lipids, such lipids or their functional groups or atoms are positively charged at a certain pH. In some embodiments, ionizable amino lipids are positively charged at acidic pH. In some embodiments, ionizable amino lipids are primarily neutral at physiological pH values ​​(e.g., about 7.0-7.4 in some examples), but positively charged at lower pH values. In some embodiments, the pKa of ionizable amino lipids can be in the range of about 5 to about 7.

[0086] As used herein, the term "isolated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from their natural coexisting material are "isolated." Individual nucleic acids or proteins can exist in substantially purified forms.

[0087] As used herein, the term "subtype" refers to a version of a molecule (e.g., a protein) that differs only slightly from another subtype of the same protein. In some embodiments, subtypes may be generated by different but related genes, or in some embodiments, they may be generated from the same gene by alternative splicing. In some embodiments, subtypes are caused by a single nucleotide polymorphism.

[0088] As used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and an RNA molecule. In some embodiments, the RNA lipid nanoparticle comprises at least one ionizable amino lipid. In some embodiments, the RNA lipid nanoparticle comprises at least one ionizable amino lipid, at least one auxiliary lipid, and at least one polymer-conjugated lipid (e.g., a PEG-conjugated lipid). In various embodiments, the average size (e.g., Z-average) of the RNA lipid nanoparticles as described herein may be from about 100 nm to 1000 nm, or from about 200 nm to 900 nm, or from about 200 nm to 800 nm, or from about 250 nm to about 700 nm. In some embodiments of this disclosure, the particle size (e.g., Z-mean) of the RNA lipid nanoparticles can be about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the average size of the lipid nanoparticles is determined by measuring the particle diameter. In some embodiments, the RNA lipid nanoparticles can be prepared by mixing lipids with the RNA molecules described herein.

[0089] As used herein, the term "lipid" refers to a lipid-like molecule. In some embodiments, lipids are amphiphilic molecules having one or more lipid-like physical properties. In the context of this disclosure, the term lipid is considered to encompass lipids.

[0090] As used herein, the term "nanoparticle" refers to a particle having an average size suitable for parenteral administration. In some embodiments, the longest dimension (e.g., diameter) of the nanoparticle is less than 1,000 nanometers (nm). In some embodiments, the nanoparticle may be characterized by a longest dimension (e.g., diameter) of less than 300 nm. In some embodiments, the nanoparticle may be characterized by a longest dimension (e.g., diameter) of less than 100 nm. In many embodiments, the nanoparticle may be characterized by a longest dimension of about 1 nm to about 100 nm, or about 1 μm to about 500 nm, or about 1 nm to 1,000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) of less than about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm, and is generally greater than about 1 nm. In many embodiments, the nanoparticle may be substantially spherical, such that its longest dimension is likely its diameter. In some embodiments, the diameter of the nanoparticle is less than 100 nm, as defined by the National Institutes of Health.

[0091] As used herein, the term "naturally occurring" means an entity that can be found in nature. For example, peptides or nucleic acids that are present in organisms (including viruses) and can be isolated from natural sources and are not intentionally modified in a laboratory are naturally occurring.

[0092] As used herein, the term "neutralization" refers to an event in which a binder (such as an antibody) binds to a biologically active site of the virus (such as a receptor-binding protein), thereby inhibiting parasitic infection of the cell. In some embodiments, the term "neutralization" refers to an event in which the binder eliminates or significantly reduces the ability of the cell to infect it.

[0093] As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single-stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both a single-stranded portion and a double-stranded portion. In some embodiments, a nucleic acid comprises a backbone containing one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone containing both phosphodiester bonds and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid may comprise a backbone containing one or more thiophosphate or 5'-N-phosphamide bonds and / or one or more peptide bonds, for example, as in "peptide nucleic acid". In some embodiments, a nucleic acid comprises one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more or all of the non-natural residues. In some embodiments, the non-natural residues comprise nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazoguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolating from a natural source, by enzymatic synthesis (e.g., by polymerization based on a complementary template, such as in vivo or in vitro, or by propagation in recombinant cells or systems), or by chemical synthesis.In some embodiments, the number of nucleic acids is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, or 140. 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0094] As used herein, the term "nucleotide" means as it is known in the art. When the number of nucleotides is used as an indicator of the size of, for example, a polynucleotide, the specific number of nucleotides refers to the number of nucleotides on a single strand of, for example, a polynucleotide.

[0095] As used herein, the term "patient" means any organism that has a disease or condition or symptom or is at risk of having said disease or condition or symptom. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient has or is susceptible to one or more diseases or conditions or symptoms. In some embodiments, the patient exhibits one or more symptoms of a disease or condition or symptom. In some embodiments, the patient has been diagnosed with one or more diseases or conditions or symptoms. In some embodiments, the disease or condition or symptom that can be treated by the provided techniques is or includes HSV infection. In some embodiments, the patient is receiving or has received some form of therapy to diagnose and / or treat a disease, condition or symptom. In some embodiments, the patient is a patient who has or is susceptible to HSV infection.

[0096] As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration as a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for parenteral administration, such as by subcutaneous, intramuscular, or intravenous injection, for example, as a sterile solution or suspension formulation.

[0097] As used herein, the terms "pharmaceuticalally effective amount" or "therapeutically effective amount" refer to the amount, alone or in combination with other doses, that achieves a desired response or expected effect. In the case of treating a specific disease, in some embodiments, the desired response relates to inhibiting the progression of the disease. In some embodiments, such inhibition may comprise slowing the progression of the disease and / or interrupting or reversing the progression of the disease. In some embodiments, the desired response in treating a disease may be or comprise delaying or preventing the onset of the disease or symptom. The effective amount of the pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) will depend on factors such as the disease or symptom to be treated, the severity of such disease or symptom, individual patient parameters including, for example, age, physical condition, size and weight, duration of treatment, type of concomitant therapy (if present), specific route of administration, and similar factors. Therefore, the dosage of the pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) may depend on a variety of such parameters. In cases where the patient's response to the initial dose is insufficient, a higher dose (or an effective higher dose achieved through a different, more localized route of administration) may be used.

[0098] As used herein, the term "poly(A) sequence" or "polyA tail" refers to a sequence of consecutive or interrupted adenosine residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3' UTR in the RNA described herein. A continuous poly(A) sequence is characterized by consecutive adenosine residues. Continuous poly(A) sequences are typical in nature. The RNA disclosed herein may have a free 3' poly(A) sequence attached to the RNA post-transcriptionally by a template-dependent RNA polymerase or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0099] As used herein, when used in connection with the occurrence of a disease, condition, and / or symptom, the term "prevent" or "prevention" means reducing the risk of developing a disease, condition, and / or symptom and / or delaying the onset of one or more characteristics or symptoms of a disease, condition, or symptom. Prevention is considered complete when the onset of a disease, condition, or symptom has been delayed for a predetermined period of time.

[0100] As used herein, the term "makexin interference domain" refers to a domain that blocks or inhibits the binding of host C3b molecules to host makexin molecules. In another embodiment, the term refers to a domain that blocks or inhibits the interaction between host C3b molecules and host makexin molecules.

[0101] As used herein, in the context of this disclosure, the term “recombinant” means “prepared through genetic engineering.” In some embodiments, in the context of this disclosure, “recombinant” entities (such as recombinant nucleic acids) are not naturally occurring.

[0102] As used herein, the term "reference" describes a standard or control relative to which comparison is made. For example, in some embodiments, the agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally contained in a tangible medium. Typically, as those skilled in the art will understand, the reference or control is determined or characterized under conditions or conditions equivalent to those of the conditions or environment being evaluated. Those skilled in the art will understand when sufficient similarity exists to justify dependence on and / or comparison with a particular possible reference or control.

[0103] As will be understood from the context, the “risk” of a disease, condition, and / or symptom refers to the likelihood that a particular individual will develop the disease, condition, and / or symptom. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, risk is expressed as risk relative to the risk associated with a reference sample or reference sample group. In some embodiments, the reference sample or reference sample group has a known risk of a disease, condition, symptom, and / or event. In some embodiments, the reference sample or reference sample group is derived from individuals comparable to the particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic traits, for example, that may predispose an individual to have (or not have) a particular disease, condition, and / or symptom. In some embodiments, risk may reflect one or more epigenetic events or traits and / or one or more lifestyle or environmental events or traits.

[0104] The terms “selectivity” or “specificity,” when used herein to refer to an active agent, are understood by those skilled in the art to mean an agent that distinguishes potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it preferentially binds to one or more competing alternative targets in the presence of such targets. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitopes, clefts, binding sites). It should be understood that specificity is not necessarily absolute. In some embodiments, specificity may be evaluated relative to the target-binding portion against the specificity of one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding portion. In some embodiments, specificity is evaluated relative to the specificity of a reference non-specific binding portion.

[0105] As used herein, the term "subject" refers to an organism to which the compositions described herein are administered, for example, for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject has a disease, condition, or symptom (e.g., HSV infection). In some embodiments, the subject is susceptible to a disease, condition, or symptom (e.g., HSV infection). In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, condition, or symptom (e.g., HSV infection). In some embodiments, the subject exhibits one or more nonspecific symptoms of a disease, condition, or symptom (e.g., HSV infection). In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, condition, or symptom (e.g., HSV infection). In some embodiments, the subject is a person having one or more characteristics that constitute susceptibility or risk to a disease, condition, or symptom (e.g., HSV infection). In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnostic and / or therapeutic treatments have been administered.

[0106] As used in this article, an individual who “has” a disease, condition, and / or symptom has been diagnosed with and / or exhibits one or more symptoms of a disease, condition, and / or symptom.

[0107] As used herein, an individual who is “vulnerable” to a disease, condition, and / or symptom is an individual who has a higher risk of developing a disease, condition, and / or symptom than members of the general population. In some embodiments, an individual who is vulnerable to a disease, condition, and / or symptom may not have been diagnosed with said disease, condition, and / or symptom. In some embodiments, an individual who is vulnerable to a disease, condition, and / or symptom may exhibit symptoms of the disease, condition, and / or symptom. In some embodiments, an individual who is vulnerable to a disease, condition, and / or symptom may not exhibit symptoms of the disease, condition, and / or symptom. In some embodiments, an individual who is vulnerable to a disease, condition, and / or symptom will develop the disease, condition, and / or symptom. In some embodiments, an individual who is vulnerable to a disease, condition, and / or symptom will not develop the disease, condition, and / or symptom.

[0108] As used herein, the term "synthetic" refers to an entity that is artificially created, prepared through human intervention, or synthesized rather than naturally occurring. For example, in some embodiments, synthetic nucleic acid or polynucleotide refers to chemical synthesis, such as nucleic acid molecules synthesized in a solid phase in some embodiments. In some embodiments, the term "synthetic" refers to an entity prepared outside of biological cells. For example, in some embodiments, synthetic nucleic acid or polynucleotide refers to nucleic acid molecules (e.g., RNA) produced by in vitro transcription using a template.

[0109] As used herein, the term "therapy" means the administration or delivery of a pharmaceutical agent or intervention that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect (e.g., one that has been shown to be statistically likely to have such an effect when administered in a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to reduce, improve, alleviate, suppress, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or features of a disease, condition, and / or symptom. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation therapy, phototherapy) that can be used to reduce, alleviate, suppress, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or features of a disease, condition, and / or symptom.

[0110] As used herein, the term "3' UTR" refers to a sequence of RNA molecules that begins after a stop codon in the coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon in the coding region of the open reading frame sequence, for example, in its natural environment. In other embodiments, the 3' UTR does not begin immediately after a stop codon in the coding region of the open reading frame sequence, for example, in its natural environment.

[0111] As used herein, the term "threshold level" refers to a level used as a reference to obtain information about the results of a measurement, such as those obtained in an assay, and / or to classify those results. For example, in some embodiments, a threshold level means a value measured in an assay that defines a boundary between two subsets of a population (e.g., batches that meet quality control standards compared to batches that do not). Thus, values ​​equal to or higher than the threshold level define one subset of the population, and values ​​lower than the threshold level define another subset of the population. The threshold level may be determined based on one or more control samples or a population of control samples. The threshold level may be determined before, during, or after the measurement of interest. In some embodiments, the threshold level may be a range of values.

[0112] As used herein, the terms “treat,” “treatment,” or “treating” mean any method used to partially or completely reduce, improve, alleviate, suppress, prevent, delay the onset of, reduce the severity of, and / or decrease the incidence of one or more symptoms or features of a disease, condition, and / or symptom. Treatment may be administered to subjects who do not exhibit signs of a disease, condition, and / or symptom. In some embodiments, treatment may be administered to subjects who exhibit only early signs of a disease, condition, and / or symptom, for example, to reduce the risk of developing a pathology associated with the disease, condition, and / or symptom. In some embodiments, treatment may be administered to subjects who are in a later stage of a disease, condition, and / or symptom.

[0113] As used herein, the term "truncated" refers to a protein or polypeptide that is shorter at the N-terminus, C-terminus, or both N-terminus and C-terminus compared to the wild-type protein or polypeptide.

[0114] As used herein, the term "vaccination" refers to the administration of a composition designed to elicit an immune response, for example, against a disease-related (e.g., pathogenic) agent. In some embodiments, vaccination may be administered before, during, and / or after exposure to the disease-related agent, and in some embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination comprises administering the vaccine composition multiple times at appropriate time intervals. In some embodiments, vaccination elicits an immune response against an infectious agent.

[0115] As used herein, the term "vaccine" refers to a composition or combination of substances or agents that, when administered to an individual, stimulate the production of antibodies or cellular immunity against a pathogen, but do not cause a serious infection. In some embodiments, the vaccine does not cause a serious infection in a subject.

[0116] As used herein in the context of molecules (e.g., nucleic acids, proteins, or small molecules), the term "variant" refers to a molecule that, compared to a reference molecule, exhibits significant structural identity with the reference molecule but is structurally different from it, for example, in the presence or absence of one or more chemical motifs or at the level of said one or more chemical motifs. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity between said particular molecule and the reference molecule. As those skilled in the art will understand, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one respect. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid due to one or more differences in the amino acid or nucleotide sequence and / or (e.g., differences in one or more chemical motifs attached to the polypeptide or nucleic acid backbone as covalent components of the polypeptide or nucleic acid, such as carbohydrates, lipids, phosphate groups). In some embodiments, the variant peptide or nucleic acid exhibits an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% with a reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid does not share at least one characteristic sequence element with the reference peptide or nucleic acid. In some embodiments, the reference peptide or nucleic acid has one or more biological activities. In some embodiments, the variant peptide or nucleic acid shares one or more of the biological activities of the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid lacks one or more of the biological activities of the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference peptide or nucleic acid. In some embodiments, if the amino acid or nucleotide sequence of the peptide or nucleic acid of interest is identical to that of a reference amino acid or nucleotide sequence, but with minor sequence changes at specific positions, the peptide or nucleic acid is considered a “variant” of the reference peptide or nucleic acid. Typically, compared to the reference, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of residues in the variant are substituted, inserted, or deleted. In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference. Typically, relative to the reference, the variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in specific biological activities).In some embodiments, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 addition or deletion compared to the reference, and in some embodiments, no addition or deletion is included. In some embodiments, the variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, or about 6 additions or deletions compared to the reference, and typically less than about 5, about 4, about 3, or about 2 additions or deletions. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature.

[0117] In some embodiments, the variant may be a sequence-conserved variant. In some embodiments, the variant may be a functionally conserved variant. In some embodiments, the variant may include the addition, deletion, or substitution of one or more amino acids.

[0118] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA loop to which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episome mammalian vectors). Other vectors (e.g., non-epithelial mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "expression vectors." In some embodiments, known techniques may be used, such as those for the generation or manipulation of recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), which is incorporated herein by reference for any purpose.

[0119] All references and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, monographs, and web pages, regardless of their form, are expressly incorporated herein by reference in their entirety. In the event of any discrepancy or conflict between one or more of the incorporated references and similar materials and this application (including but not limited to definitions of terminology, usage of terms, and described techniques), this application shall prevail. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way.

[0120] Polynucleotides encoding HSV polypeptides

[0121] In some embodiments, this disclosure provides a polynucleotide encoding a polypeptide comprising a viral antigen from herpes simplex virus (HSV) or an immunogenic fragment thereof. In some embodiments, this disclosure provides a nucleoside-modified polynucleotide encoding a polypeptide comprising a viral antigen from herpes simplex virus (HSV) or an immunogenic fragment thereof. In some embodiments, the term "polynucleotide" as used herein may be used interchangeably with "RNA". In some embodiments, the HSV viral antigen or an immunogenic fragment thereof is derived from HSV-2. In another embodiment, the HSV viral antigen or an immunogenic fragment thereof is derived from HSV-1. Therefore, in some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding a polypeptide comprising a viral antigen from herpes simplex virus-2 (HSV-2) or an immunogenic fragment thereof.

[0122] HSV glycoprotein B

[0123] In some embodiments, this disclosure provides a polynucleotide or RNA encoding a polypeptide comprising glycoprotein B (gB) of HSV or an immunogenic fragment thereof. In some embodiments, the polynucleotide or RNA encodes a polypeptide comprising gB (gB1) of HSV-1 or an immunogenic fragment thereof. In another embodiment, the polynucleotide or RNA encodes a polypeptide comprising gB (gB2) of HSV-2 or an immunogenic fragment thereof.

[0124] In some embodiments, this disclosure provides a nucleoside-modified polynucleotide or RNA encoding a polypeptide comprising HSV glycoprotein B (gB) or an immunogenic fragment thereof. In some embodiments, the nucleoside-modified polynucleotide or RNA encodes a polypeptide comprising HSV-1 gB (gB1) or an immunogenic fragment thereof. In another embodiment, the nucleoside-modified polynucleotide or RNA encodes a polypeptide comprising HSV-2 gB (gB2) or an immunogenic fragment thereof.

[0125] In some embodiments, this disclosure provides an RNA encoding a polypeptide comprising full-length HSV-1 gB. In some embodiments, this disclosure provides a nucleoside-modified RNA encoding a polypeptide comprising full-length HSV-1 gB. In some embodiments, the amino acid sequence of the full-length HSV-1 gB comprises the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 11. In some embodiments, the HSV-1 gB fragment comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11. In another embodiment, the amino acid sequence of the full-length HSV-1 gB sequence comprises the sequence shown in SEQ ID NO: 9. In some embodiments, the nucleotide sequence of HSV-1 gB comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as SEQ ID NO: 9.

[0126] In another embodiment, the amino acid sequence of the HSV-1 gB antigen sequence comprises the sequence shown in SEQ ID NO: 199. In some embodiments, the amino acid sequence of the HSV-1 gB antigen is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 199. In another embodiment, the amino acid sequence of the HSV-1 gB antigen sequence comprises the sequence shown in SEQ ID NO: 200. In some embodiments, the amino acid sequence of the HSV-1 gB antigen is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 200.

[0127] In some embodiments, this disclosure provides an RNA encoding a polypeptide comprising full-length HSV-2 gB. In some embodiments, this disclosure provides an RNA encoding a polypeptide or a nucleoside-modified RNA comprising full-length HSV-2 gB. In some embodiments, the amino acid sequence of the full-length HSV-2 gB comprises the sequence shown in SEQ ID NO: 7. In some embodiments, the HSV-1 gB fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 7.

[0128] In another embodiment, the amino acid sequence of the HSV-2 gB antigen sequence comprises the sequence shown in SEQ ID NO: 201. In some embodiments, the amino acid sequence of the HSV-2 gB antigen is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 201. In another embodiment, the amino acid sequence of the HSV-2 gB antigen sequence comprises the sequence shown in SEQ ID NO: 202. In some embodiments, the amino acid sequence of the HSV-2 gB antigen is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to that of SEQ ID NO: 202.

[0129] In some embodiments, this disclosure provides an RNA encoding a polypeptide or a nucleoside-modified RNA, said polypeptide comprising an extracellular domain of HSV-1 gB or an immunogenic fragment thereof. In some embodiments, the extracellular domain of HSV-1 gB is as shown in SEQ ID NO: 10.

[0130] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB comprises SEQ ID NO: 203. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0131] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB comprises SEQ ID NO: 204. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 204.

[0132] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB comprises SEQ ID NO: 205. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 205.

[0133] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB comprises SEQ ID NO: 206. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 206.

[0134] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB comprises SEQ ID NO: 207. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-1 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 207.

[0135] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 208. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 208.

[0136] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 209. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 209.

[0137] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 210. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 210.

[0138] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 211. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211.

[0139] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 212. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 212.

[0140] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 213. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 213.

[0141] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 214. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 214.

[0142] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 215. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 215.

[0143] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 216. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 216.

[0144] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 217. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 217.

[0145] In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA comprises SEQ ID NO: 218. In some embodiments, the RNA encoding at least the immunogenic portion of HSV-1 gB or the nucleoside-modified RNA is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 218.

[0146] In another embodiment, this disclosure provides an RNA or nucleoside-modified RNA encoding a polypeptide comprising an extracellular domain of HSV-2 gB or an immunogenic fragment thereof. In some embodiments, the extracellular domain of HSV-2 gB comprises an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the extracellular domain of HSV-2 gB consists of or is substantially composed of the amino acid sequence of SEQ ID NO: 1. In some embodiments, this disclosure covers a sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1. In some embodiments, the extracellular domain of HSV-2 gB is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 5. In some embodiments, the extracellular domain of HSV-2 gB is encoded by a nucleotide sequence consisting of or substantially composed of SEQ ID NO: 5. In some embodiments, this disclosure covers a sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 5.

[0147] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 219. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 219.

[0148] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 220. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 220.

[0149] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 221. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221.

[0150] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 222. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 222.

[0151] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB comprises SEQ ID NO: 223. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 223.

[0152] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB comprises SEQ ID NO: 224. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 224.

[0153] In some embodiments, this disclosure provides an RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB comprises SEQ ID NO: 225. In some embodiments, the RNA or nucleoside-modified RNA encoding at least an immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225.

[0154] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 226. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 226.

[0155] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 227. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 227.

[0156] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 228. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 228.

[0157] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 229. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 229.

[0158] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB comprises SEQ ID NO: 230. In some embodiments, the RNA or nucleoside-modified RNA encoding at least the immunogenic portion of HSV-2 gB is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 230.

[0159] Other HSV glycoproteins

[0160] In some embodiments, in addition to RNA encoding HSV gB or its immunogenic fragment or nucleoside-modified RNA, the compositions disclosed herein may further comprise one or more RNAs encoding HSV glycoprotein C (gC) or its immunogenic fragment, HSV glycoprotein D (gD) or its immunogenic fragment, HSV glycoprotein E (gE) or its immunogenic fragment, HSV glycoprotein H (gH) or its immunogenic fragment, HSV glycoprotein L (gL) or its immunogenic fragment, HSV glycoprotein I (gI) or its immunogenic fragment, or any combination thereof.

[0161] HSV glycoprotein C

[0162] In some embodiments, the compositions disclosed herein further comprise RNA encoding a polypeptide or nucleoside-modified RNA, said polypeptide comprising or consisting of HSV glycoprotein C (gC) or an immunogenic fragment thereof.

[0163] In some embodiments, this document discloses a single composition comprising RNA encoding HSV gB and RNA encoding HSV gC or an immunogenic fragment thereof. In some embodiments, the gB and gC RNAs are portions of a single polynucleotide. In some embodiments, this document discloses two separate compositions to be administered as part of a single treatment, a first composition comprising RNA encoding HSV gB or an immunogenic fragment thereof, and a second composition comprising RNA encoding HSV gC or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gC or an immunogenic fragment thereof comprises RNA encoding HSV-1 gC (also referred to as gC1) or an immunogenic fragment thereof. In some embodiments, the nucleotide sequence of the RNA encoding the HSV-1 gC fragment comprises the following:

[0164]

[0165]

[0166] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent the 5' untranslated sequence (SEQ ID NO: 13). In some embodiments, bolded residues represent the signal sequence (leader sequence) (SEQ ID NO: 14). In some embodiments, italicized residues represent the 3' untranslated sequence (SEQ ID NO: 15) and the polyadenylated tail (SEQ ID NO: 16).

[0167] In some embodiments, the polynucleotide encoding the HSV-1 gC signal sequence MHCII includes a sequence

[0168] ATGGCCATCTCCGGCGTGCCCGTGCTGGGCTTCTTCATCATCGCCGTGCTGATGTCCGCCCAGGAGTCCTGGGCC (SEQ ID NO: 14).

[0169] In some embodiments, the 3' UTR contains a sequence

[0170] CTAGTAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCT (SEQ ID NO: 15).

[0171] In another embodiment, the nucleotide sequence of the RNA encoding the HSV-1 gC fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof. In some embodiments, the sequence of the HSV-1 gC fragment is as shown in SEQ ID NO: 17.

[0172] In some embodiments, the HSV-1 gC fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 27-457 from the gC of HSV-1 (e.g., KOS strain), as shown in the following amino acid sequence:

[0173] ETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMYY LAWGRMDSPHEYGTWVRVRMFRPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWH RDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHH (SEQ ID NO: 18).

[0174] In some embodiments, the HSV-1 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 18. In some embodiments, the HSV-1 gC fragment has the same amino acid sequence as SEQ ID NO: 18. In some embodiments, the gC fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 27-457 of the gC from the HSV-1 strain.

[0175] In some embodiments, the HSV-1 gC fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 25-457 from the gC of HSV-1 (e.g., KOS strain), as shown in the following amino acid sequence:

[0176] GSETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMY YLAWGRMDSPHEYGTWVRVRMFRPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTW HRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHH (SEQ ID NO: 19).

[0177] In some embodiments, the HSV-1 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 19. In some embodiments, the HSV-1 gC fragment has the same amino acid sequence as SEQ ID NO: 19.

[0178] In some embodiments, the full-length HSV-1 gC encoded by the RNA used in the methods and compositions of this disclosure comprises the following amino acid sequence:

[0179] MAPGRVGLAVVLWGLLWLGAGVAGGSETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCD RRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMYYLAWGRMDSPHEYGT WVRVRMFRPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVC TAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHHGSHQPPPRDPTERQVIEAIEWVGIGIGVLAAGVLVVTAIVYVVRTSQSRQRHRR (SEQ ID NO:20).

[0180] In some embodiments, HSV-1 gC comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 20. In some embodiments, HSV-1 gC has the same amino acid sequence as SEQ ID NO: 20.

[0181] In other embodiments, the HSV-1 gC or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises an amino acid sequence as shown in any of the following GenBank accession numbers: AAA45779.1, AAA96680.1, ABI63505.1, ABM52973.1, ABM52976.1, ABM52977.1, ACM62267.1, ADD60042.1, ADD60119.1, ADM22367.1, ADM22444.1, ADM22520.1, ADM22597.1, ADM22674.1, ADM22751.1, ADM22827.1, ADM22904.1, ADM22981.1, A DM23057.1, ADM23133.1, ADM23210.1, ADM23287.1, ADM23361.1, ADM23435.1, ADM23509.1, ADM23583.1, ADM23658.1, ADM23733.1, ADM23809.1, AEQ77075.1, AEQ77099.1, AER37628.1, AER37697.1, AER37767.1, AER37838.1, AER37910.1, AER37981.1, AER38051.2, AFA36179.1, AFA36180.1, AFA36181.1, AFA36182.1, AFA36183.1, AFA36184.1, AFA36185.1, AFA36186.1, AFA36187.1, AFA36188.1, AFA36189.1, AFA36190.1, AFA36191.1 , AFA36192.1, AFA36193.1, AFA36194.1, AFA36195.1, AFA36196.1, AFA36197.1, AFA36198.1, AFA36199.1, AFA36200.1, AFA36201.1, AFA36202.1 , AFA36203.1, AFE62872.1, AFH78104.1, AFI23635.1, AFK50391.1, AFP86408.1, AGZ01906.1, AIR95840.1, AJE59989.1, AJE60060.1, AJE60131. 1. AJE60202.1, AKE48623.1, AKE98415.1, AKE98416.1, AKE98417.1, AKE98418.1, AKE98419.1, AKE98420.1, AKE98421.1, AKE98422.1, AKE98423.1、AKE98424.1、AKE98425.1、AKE98426.1、AKE98427.1、AKE98428.1、AKE98 429.1、AKE98430.1、AKE98431.1、AKE98432.1、AKE98433.1、AKE98434.1、AK E98435.1, AKG59227.1, AKG59299.1, AKG59372.1, AKG59444.1, AKG59516.1, AKG59591.1, AKG59663.1, AKG59736.1, AKG59807.1, AKG59879.1, AKG599 53.1, AKG60027.1, AKG60099.1, AKG60170.1, AKG60243.1, AKG60316.1, AKG60386.1, AKG60456.1, AKG60528.1, AKG60601.1, AKG60674.1, AKG60745.1 、AKG60817.1、AKG60887.1、AKG60959.1、AKG61032.1、AKG61104.1、AKG611 75.1、AKG61248.1、AKG61321.1、AKG61392.1、AKG61464.1、AKG61537.1、AKG 61611.1, AKG61684.1, AKG61756.1, AKG61828.1, AKG61902.1, AKG61974.1, AKH80444.1, AKH80517.1, AKM76368.1, ALM22613.1, ALM22687.1, ALM2276 1.1、ALM22835.1、ALO18641.1、ALO18717.1、AMB65642.1、AMB65715.1、AMB 65862.1、AMN09813.1、ANN83942.1、ANN84019.1、ANN84095.1、ANN84172.1、 ANN84249.1、ANN84326.1、ANN84403.1、ANN84478.1、ANN84555.1、ANN8463 2.1、ANN84708.1、ANN84785.1、ANN84861.1、ANN84938.1、ANN85014.1、ANN8 5091.1, ANN85167.1, ANN85242.1, ANN85319.1, ANN85396.1, ANN85472.1, ANN85549.1, ANN85626.1, ANN85703.1, ANN85779.1, AOY34308.1, AOY36663.1. AOY36687.1, ARB08935.1, ARO38059.1, ARO38060.1, ARO38061.1, ARO38062.1, ARO38063.1, ARO3806 4.1, ARO38065.1, ARO38066.1, ASM47642.1, ASM47719.1, ASM47796.1, ASM47871.1, BAM73394.1, CAA322 94.1, CAB40083.1, CAD13356.1, CAD13357.1, CAD13358.1, CAD13359.1, CAD13360.1, CAD13361.1, CAD1 3362.1, CAD13363.1, CAD13364.1, CAD13365.1, CAD13366.1, CAD13367.1, CAD13368.1, CAD13369.1, CAD 13370.1, CAD13371.1, CAD13372.1, CAD13373.1, CAD13374.1, CAD13375.1, CAD13376.1, CAD13377.1, C AD13378.1, P04290.1, P04488.1, P09855.1, P10228.1, P28986.1, SBO07729.1, SBO07793.1, SBO07798.1 , SBO07812.1, SBO07880.1, SBS69375.1, SBS69379.1, SBS69440.1, SBS69448.1, SBS69560.1, SBS69599. 1. SBS69602.1, SBS69637.1, SBS69790.1, SBT69374.1, SCL76887.1, YP_009137119.1 or YP_009137143.1. .

[0182] In some embodiments, the RNA encoding HSV gC as described herein comprises RNA encoding HSV-2 gC (also known as gC2) or an immunogenic fragment thereof.

[0183] In some embodiments, the nucleotide sequence of the RNA encoding the HSV-2 gC fragment comprises the following:

[0184]

[0185]

[0186] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent the 5' untranslated sequence (SEQ ID NO: 13). In some embodiments, bolded residues represent the signal sequence (leader sequence) (SEQ ID NO: 3). In some embodiments, italicized residues represent the 3' untranslated sequence (SEQ ID NO: 15) and the polyadenylated tail (SEQ ID NO: 16).

[0187] In another embodiment, the nucleotide sequence of the RNA encoding the HSV-2 gC fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof. In some embodiments, the sequence of the HSV-2 gC fragment is as shown in SEQ ID NO: 22.

[0188] In some embodiments, the HSV-2 gC fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 27-426 from the gC of HSV-2 (e.g., strain 333 or UL44), as shown in the following amino acid sequence:

[0189] ASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTESRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWV RVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 23).

[0190] In some embodiments, the HSV-2 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 23. In some embodiments, the HSV-2 gC fragment has the same amino acid sequence as SEQ ID NO: 23.

[0191] In some embodiments, the HSV-2 gC fragment comprises the following amino acid sequence:

[0192] ASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWV RVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 24).

[0193] In some embodiments, the HSV-2 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 24. In some embodiments, the HSV-2 gC fragment has the same amino acid sequence as SEQ ID NO: 24.

[0194] In some embodiments, the HSV-2 gC fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 28-426 from the gC of HSV-2 (e.g., strain 333 or UL44), as shown in the following amino acid sequence:

[0195] SPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTESRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWV RVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 25).

[0196] In some embodiments, the HSV-2 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 25. In some embodiments, the HSV-2 gC fragment has the same amino acid sequence as SEQ ID NO: 25.

[0197] In some embodiments, the HSV-2 gC fragment comprises the following amino acid sequence:

[0198] SPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWV RVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 26).

[0199] In some embodiments, the HSV-2 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 26. In some embodiments, the HSV-2 gC fragment has the same amino acid sequence as SEQ ID NO: 26.

[0200] In some embodiments, the HSV-2 gC fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 26-426 from the gC of HSV-2 (e.g., strain 333 or UL44), as shown in the following amino acid sequence:

[0201] SASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTESRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTW VRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 27).

[0202] In some embodiments, the HSV-2 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 27. In some embodiments, the HSV-2 gC fragment has the same amino acid sequence as SEQ ID NO: 27.

[0203] In some embodiments, the HSV-2 gC fragment comprises the following amino acid sequence:

[0204] SASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTW VRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 28).

[0205] In some embodiments, the HSV-2 gC fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 28. In some embodiments, the HSV-2 gC fragment has the same amino acid sequence as SEQ ID NO: 28.

[0206] In some embodiments, the full-length HSV-2 gC encoded by the RNA used in the methods and compositions of this disclosure comprises the following amino acid sequence:

[0207] MALGRVGLAVGLWGLLWVGVVVVLANASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRT EFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHP HAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEG VTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHHGSHQPPPRDPTERQVIRAVEGAGIGVAVLVAVVLAGTAVVYLTHASSVRYRRLR (SEQ ID NO: 29).

[0208] In some embodiments, HSV-2 gC comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 29. In some embodiments, HSV-2 gC has the same amino acid sequence as SEQ ID NO: 29.

[0209] In another embodiment, the HSV-2 gC or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises an amino acid sequence as shown in any of the following GenBank accession numbers: AAA20532.1, AAA66442.1, AAB60549.1, AAB60550.1, AAB60551.1, AAB72101.1, ABU45429.1, ABU45430.1, ABU45431.1, ABU45432.1, ABU45459.1, ABU45460.1, AEV91348.1, AEV91383.1, AEV91407.1, AFM93864.1, AHG54708.1, AKC42808.1, A KC59285.1, AKC59357.1, AKC59428.1, AKC59499.1, AKC59570.1, AMB66008.1, AMB66079.1, AMB66151.1, AMB66224.1, AMB66252.1, AMB66253.1, AMB66368.1, AMB66441.1, AQZ55735.2, AQZ55806.1, AQZ55877.1, AQZ55 948.1, AQZ56019.1, AQZ56090.1, AQZ56161.2, AQZ56232.2, AQZ56303.2, AQZ56374.2, AQZ56445.1, AQZ56516.1, AQZ56587.1, AQZ56658.1, AQZ56 729.2, AQZ56800.1, AQZ56871.1, AQZ56942.2, AQZ57013.1, AQZ57084.2 , AQZ57155.1, AQZ57226.1, AQZ57297.1, AQZ57368.1, AQZ57439.1, AQZ5 7510.1, AQZ57581.1, AQZ57652.1, AQZ57723.1, AQZ57794.2, AQZ57865.2 , AQZ57936.1, AQZ58007.2, AQZ58078.1, AQZ58149.2, AQZ58220.1, AQZ5 8291.1, AQZ58362.1, AQZ58433.1, AQZ58504.1, AQZ58575.1, AQZ58646. 1. AQZ58717.2, AQZ58788.2, AQZ58859.2, AQZ58930.1, AQZ59001.2, AQZ 59072.1, AQZ59143.1, ARO38067.1, ARO38068.1, ARO38069.1, ARO38070.1. ARO38071.1, ARO38072.1, CAA25687.1, CAA26025.1, CAB06730.1, CAB06734.1, CAB96544.1, P 03173.1, P06475.1, P89475.1, Q89730.1, YP_009137161.1, YP_009137196.1 or YP_009137220.1. .

[0210] In some embodiments, the gC protein fragment encoded by the RNA used in the methods and compositions of this disclosure includes a complement interference domain.

[0211] HSV glycoprotein D

[0212] In some embodiments, the compositions disclosed herein further comprise RNA encoding a polypeptide or nucleoside-modified RNA, said polypeptide comprising or consisting of HSV glycoprotein D (gD) or an immunogenic fragment thereof.

[0213] In some embodiments, this document discloses a single composition comprising RNA encoding HSV gB and RNA encoding HSV gD or an immunogenic fragment thereof. In some embodiments, the gB and gD RNAs are portions of a single polynucleotide. In some embodiments, this document discloses two separate compositions to be administered as part of a single treatment, a first composition comprising RNA encoding HSV gB or an immunogenic fragment thereof, and a second composition comprising RNA encoding HSV gD or an immunogenic fragment thereof.

[0214] In some embodiments, the RNA encoding HSV gD or an immunogenic fragment thereof comprises RNA encoding HSV-1 gD (also known as gD1) or an immunogenic fragment thereof. In some embodiments, the nucleotide sequence of the RNA encoding the HSV-1 gD fragment comprises the following:

[0215]

[0216]

[0217] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent the 5' untranslated sequence (SEQ ID NO: 13). In some embodiments, bolded residues represent the signal sequence (leader sequence) (SEQ ID NO: 38). In some embodiments, italicized residues represent the 3' untranslated sequence (SEQ ID NO: 3) and the polyadenylated tail (SEQ ID NO: 15).

[0218] In another embodiment, the nucleotide sequence of the RNA encoding the HSV-1 gD fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof. In some embodiments, the polynucleotide sequence of the HSV-1 gD fragment is as shown in SEQ ID NO: 32.

[0219] In some embodiments, the HSV-1 gD fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 26-331 of gD (e.g., from the HSV-1 Patton strain), as shown in the following amino acid sequence:

[0220] KYALADASLKMADPNRFRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHA PAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPY (SEQ ID NO: 32)

[0221] In some embodiments, the HSV-1 gD fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 32. In some embodiments, the HSV-1 gD fragment has the same amino acid sequence as SEQ ID NO: 32.

[0222] In some embodiments, the full-length HSV-1 gD encoded by the RNA used in the methods and compositions of this disclosure comprises the following amino acid sequence:

[0223] MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKLADPNRFRRKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWT EITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGLMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEAPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (SEQ ID NO: 33)

[0224] In some embodiments, HSV-1 gD comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 33. In some embodiments, HSV-1 gD has the same amino acid sequence as SEQ ID NO: 33.

[0225] In another embodiment, the HSV-1 gD or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises an amino acid sequence as shown in any of the following GenBank accession numbers: AAL90884.1 (KHS2 strain), AAL90883.1 (KHS1 strain), AAK93950.1 (F strain), AAB59754.1 (F strain), AAA19631.1 (unidentified mutant strain), AAA19630.1 (unidentified mutant strain), or AAA19629.1 (unidentified strain).

[0226] In another embodiment, the HSV-1 gD or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises an amino acid sequence as shown in any of the following GenBank accession numbers: A1Z0Q5.2, AAA45780.1, AAA45785.1, AAA45786.1, AAA96682.1, AAK19597.1, AAN74642.1, ABI63524.1, ABM52978.1, ABM52979.1, ABM52980.1, ABM52981.1, ABM66847.1, ABM66848.1, ACM62295.1, ADD60053.1, ADD60130.1, ADM22389.1, AD A A ER37786.1, AER37857.1, AER37929.1, AER38000.1, AER38070.1, AFE62894.1, AFH41180.1, AFI23657.1, AFK50415.1, AFP86430.1, AGZ01928.1, AIR95858.1, AJE60009.1, AJE60080.1, AJE60151.1, AJE60222.1, AJE60 293.1, AJE60439.1, AKE48645.1, AKG59246.1, AKG59318.1, AKG59391.1 , AKG59462.1, AKG59536.1, AKG59609.1, AKG59682.1, AKG59755.1, AKG5 9826.1, AKG59898.1, AKG59972.1, AKG60046.1, AKG60118.1, AKG60189. 1. AKG60261.1, AKG60334.1, AKG60404.1, AKG60474.1, AKG60546.1, AKG 60620.1, AKG60692.1, AKG60763.1, AKG60835.1, AKG60906.1, AKG60978.1、AKG61050.1、AKG61123.1、AKG61194.1、AKG61267.1、AKG61339.1、AKG61 411.1、AKG61484.1、AKG61556.1、AKG61629.1、AKG61703.1、AKG61774.1、AK G61847.1, AKG61920.1, AKG61993.1, AKH80463.1, AKH80536.1, ALM22635.1, ALM22709.1, ALM22783.1, ALM22857.1, ALO18662.1, ALO18738.1, AMB656 62.1, AMB65735.1, AMB65809.1, AMB65885.1, AMB65956.1, AMN09832.1, ANN83964.1, ANN84041.1, ANN84117.1, ANN84194.1, ANN84271.1, ANN84348.1 、ANN84424.1、ANN84500.1、ANN84577.1、ANN84653.1、ANN84730.1、ANN848 06.1、ANN84883.1、ANN84959.1、ANN85036.1、ANN85112.1、ANN85187.1、ANN 85264.1, ANN85341.1, ANN85416.1, ANN85494.1, ANN85571.1, ANN85648.1, ANN85724.1, ANN85801.1, AOY34093.1, AOY34141.1, AOY34243.1, AOY3427 1.1, AOY34337.1, AOY36685.1, ARB08957.1, ARO37961.1, ARO37962.1, ARO37963.1, ARO37964.1, ARO37965.1, ARO37966.1, ARO37967.1, ARO37968.1 ARO37969.1, ARO37970.1, ARO37971.1, ARO37972.1, ARO37973.1, ARO37974.1, ARO37975.1, ARO37976.1, ARO37977.1, ARO37978.1, ARO37979.1, ARO3 7980.1, ARO37981.1, ARO37982.1, ARO37983.1, ARO37984.1, ARO37985.1, ARO37986.1, ARO37987.1, ARO37988.1, ARO37989.1, ARO37990.1, ARO37991.1. ARO37992.1, ARO37993.1, ARO37994.1, ARO37995.1, ARO37996.1, ARO37997.1, ARO37998.1, ARO37999.1, ASM47664.1, ASM47741.1, ASM478 18.1, ASM47893.1, BAM73419.1, CAA26060.1, CAA32283.1, CAA32284.1, CAA32289.1, CAA38245.1, CAT05431.1, P06476.1, P36318.1, P57083.1 , P68331.1, Q05059.1, Q69091.1, SBO07792.1, SBO07819.1, SBO07855.1, SBO07869.1, SBO07887.1, SBO07908.1, SBS69553.1, SBS69561.1, SB S69579.1, SBS69625.1, SBS69688.1, SBS69694.1, SBS69717.1, SBS69727.1, SBS69811.1, SBT69395.1, SCL76902.1, VGBEDZ or YP_009137141.1. .

[0227] In some embodiments, the RNA encoding HSV gD as described herein comprises RNA encoding HSV-2 gD (also known as gD2) or an immunogenic fragment thereof.

[0228] In some embodiments, the nucleotide sequence of the RNA encoding the HSV-2 gD fragment comprises the following:

[0229]

[0230]

[0231] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent the 5' untranslated sequence (SEQ ID NO: 13). In some embodiments, bolded residues represent the signal sequence (leader sequence) (SEQ ID NO: 38). In some embodiments, italicized residues represent the 3' untranslated sequence (SEQ ID NO: 15) and the polyadenylated tail (SEQ ID NO: 16).

[0232] In some embodiments, the polynucleotide encoding the gD2 signal sequence comprises a sequence

[0233] ATGACCCGCCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCCGCGCC (SEQ ID NO: 38).

[0234] In another embodiment, the nucleotide sequence of the RNA encoding the HSV-2 gD fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof. In some embodiments, the nucleotide sequence of the RNA encoding the HSV-2 gD fragment is as shown in SEQ ID NO: 35.

[0235] In some embodiments, the HSV-2 gD fragment encoded by the RNA used in the methods and compositions of this disclosure contains amino acids 26-331 of gD (e.g., from HSV-2 strain 333 or US6), as shown in the following amino acid sequence:

[0236] KYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHA PAFETAGTYLRLVKINDWTEITQFILEHRRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHH (SEQ ID NO: 36).

[0237] In some embodiments, the HSV-2 gD fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 36. In some embodiments, the HSV-2 gD fragment has the same amino acid sequence as SEQ ID NO: 36.

[0238] In some embodiments, the HSV-2 gD fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 30-331 of gD from HSV-2 (e.g., strain 333 or US6), as shown in the following amino acid sequence:

[0239] ADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPA FETAGTYLRLVKINDWTEITQFILEHRRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHH (SEQ ID NO: 39).

[0240] In some embodiments, the HSV-2 gD fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 39. In some embodiments, the HSV-2 gD fragment has the same amino acid sequence as SEQ ID NO: 39.

[0241] In some embodiments, the HSV-2 gD fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 31-331 of gD from HSV-2 (e.g., strain 333 or US6), as shown in the following amino acid sequence:

[0242] DPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPA FETAGTYLRLVKINDWTEITQFILEHRRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHH (SEQ ID NO: 40).

[0243] In some embodiments, the HSV-2 gD fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 40. In some embodiments, the HSV-2 gD fragment has the same amino acid sequence as SEQ ID NO: 40.

[0244] In some embodiments, the full-length HSV-2 gD encoded by the RNA used in the methods and compositions of this disclosure comprises the following amino acid sequence:

[0245] MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDW TEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGLMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (SEQ ID NO: 37).

[0246] In some embodiments, HSV-2 gD comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 37. In some embodiments, HSV-2 gD has the same amino acid sequence as SEQ ID NO: 37.

[0247] In another embodiment, the HSV-2 gD or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises the amino acid sequence shown in the GenBank accession number: 1003204A, AAA45841.1, AAA45842.1, AAB60552.1, AAB60553.1, AAB60554.1, AAB60555.1, AAB72102.1, AAS01730.1, AAW23130.1, AAW23131.1, AAW23132.1, AAW23133.1, AAW23134.1, ABS84899.1, ABU45433.1, ABU45434.1, ABU45435.1, ABU45461.1, A BU45462.1, ACA28831.1, AEV91405.1, AFM93876.1, AFS18198.1, AFS18199.1, AFS18200.1, AFS18201.1, AFS18202.1, AFS18203.1, AFS18204.1, AFS18205.1, AFS18206.1, AFS18207.1, AFS18208.1, AFS18209.1, AFS18210.1, AFS18211.1, AFS18212.1, AFS18213.1, AFS18214.1, AFS18215.1, AFS18216.1, AFS18217.1, AFS18218.1, AFS18219.1, AFS18220.1, AFS18221.1, AHG54730.1, AIL27720.1, AIL27721.1, AIL27722.1, AIL27723.1 , AIL27724.1, AIL27725.1, AIL27726.1, AIL27727.1, AIL27728.1, AIL27729.1, AIL27730.1, AIL27731.1, AIL28069.1, AIL28070.1, AKC42828.1 , AKC59305.1, AKC59376.1, AKC59447.1, AKC59518.1, AKC59589.1, AMB66102.1, AMB66171.1, AMB66244.1, AMB66321.1, AMB66394.1, AMB66463. 1. AQZ55754.1, AQZ55825.1, AQZ55896.1, AQZ55967.1, AQZ56038.1, AQZ 56109.1, AQZ56180.1, AQZ56251.1, AQZ56322.1, AQZ56393.1, AQZ56464.1. AQZ56535.1, AQZ56606.1, AQZ56677.1, AQZ56748.1, AQZ56819.1, AQZ56890.1, AQZ56961.1, AQZ57032.1, AQZ57103.1, AQZ57174.1, AQZ57245.1, AQZ57316.1, AQZ57387.1, AQZ57458.1, AQZ57529.1, AQZ57600.1, AQZ57671.1, AQZ57742.1, AQZ57813.1, AQZ57884.1, AQZ57955.1, AQZ58026.1, AQZ58097.1, AQZ58168.1, AQZ58239.1, AQZ58310.1, AQZ58381.1, AQZ58452.1, AQZ58523.1, AQZ58594.1, AQZ58665.1, AQZ58736.1, AQZ58807.1, AQZ58878.1, AQZ58949.1, AQZ59020.1, AQZ59091.1, AQZ59162.1, ARO38000.1, ARO38001.1, ARO38002.1, ARO38003.1, ARO38004.1, ARO38005.1, ARO38006.1, ARO38007.1, ARO38008.1, ARO38009.1, ARO38010.1, ARO38011.1, ARO38012.1, ARO38013.1, ARO38014.1, ARO38015.1, ARO38016.1, ARO38017.1, ARO38018.1, ARO38019.1, ARO38020.1, ARO38021.1, ARO38022.1, ARO38023.1, ARO38024.1, ARO38025.1, ARO38026.1, ARO38027.1, ARO38028.1, ARO38029.1, ARO38030.1, ARO38031.1, ARO38032.1, ARO38033.1, ARO38034.1, ARO38035.1, ARO38036.1, ARO38037.1, ARO38038.1, ARO38039.1, ARO38040.1, ARO38041.1, ARO38042.1, ARO38043.1, ARO38044.1, CAA26025.1, CAB06713.1, CAC33573.1, CAT05432.1, P03172.2, Q69467.1 or YP_009137218.1.

[0248] In some embodiments, the gD protein or fragment (e.g., an immunogenic fragment) includes Y63. In another embodiment, the gD protein or fragment (e.g., an immunogenic fragment) includes R159. In another embodiment, the gD protein or fragment (e.g., an immunogenic fragment) includes D240. In another embodiment, the gD protein or fragment (e.g., an immunogenic fragment) includes P246. In another embodiment, the gD protein or fragment (e.g., an immunogenic fragment) includes residues selected from Y63, R159, D240, and P246. In another embodiment, including one of these residues induces an antibody that inhibits binding to adhesion protein-1.

[0249] HSV glycoprotein E

[0250] In some embodiments, the compositions disclosed herein further comprise RNA encoding a polypeptide or nucleoside-modified RNA, said polypeptide comprising or consisting of HSV glycoprotein E (gE) or an immunogenic fragment thereof.

[0251] In some embodiments, this document discloses a single composition comprising RNA encoding HSV gB and RNA encoding HSV gE or an immunogenic fragment thereof. In some embodiments, the gB and gE RNAs are portions of a single polynucleotide. In some embodiments, this document discloses two separate compositions to be administered as part of a single treatment, a first composition comprising RNA encoding HSV gB or an immunogenic fragment thereof, and a second composition comprising RNA encoding HSV gE or an immunogenic fragment thereof.

[0252] In some embodiments, the RNA encoding HSV gE or an immunogenic fragment thereof comprises RNA encoding HSV-1 gE (also known as gE1) or an immunogenic fragment thereof. In some embodiments, the nucleotide sequence of the RNA encoding the HSV-1 gE fragment comprises the following:

[0253]

[0254]

[0255] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent the 5' untranslated sequence (SEQ ID NO: 13). In some embodiments, bolded residues represent the signal sequence (leader sequence) (SEQ ID NO: 3). In some embodiments, italicized residues represent the 3' untranslated sequence (SEQ ID NO: 15) and the polyadenylated tail (SEQ ID NO: 16).

[0256] In another embodiment, the nucleotide sequence of the RNA encoding the HSV-1 gE fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof. In some embodiments, the sequence of the HSV-1 gE fragment is as shown in SEQ ID NO: 42.

[0257] In some embodiments, the HSV-1 gE fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 24-409 of gE from HSV-1 (e.g., NS strain), as shown in the following amino acid sequence:

[0258] KTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAPEV SHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGA (SEQ ID NO: 43).

[0259] In some embodiments, the HSV-1 gE fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 43. In some embodiments, the HSV-1 gE fragment has the same amino acid sequence as SEQ ID NO: 43. In some embodiments, the gE fragment encoded by RNA used in the methods and compositions of this disclosure comprises amino acids 24-409 of gE from an HSV-1 strain (e.g., SEQ ID NO: 43).

[0260] In some embodiments, the HSV-1 gE fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 21-409 of gE from HSV-1 (e.g., NS strain or US8), as shown in the following amino acid sequence:

[0261] GTPKTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAP EVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGA (SEQ ID NO: 44).

[0262] In some embodiments, the HSV-1 gE fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 44. In some embodiments, the HSV-1 gE fragment has the same amino acid sequence as SEQ ID NO: 44. In some embodiments, the gE fragment encoded by RNA used in the methods and compositions of this disclosure comprises amino acids 21-409 of gE from an HSV-1 strain (e.g., SEQ ID NO: 44).

[0263] In some embodiments, the full-length HSV-1 gE encoded by the RNA used in the methods and compositions of this disclosure comprises the following amino acid sequence:

[0264] (SEQ ID NO: 45).

[0265] In some embodiments, HSV-1 gE comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 45. In some embodiments, HSV-1 gE has the same amino acid sequence as SEQ ID NO: 45.

[0266] In another embodiment, the HSV-1 gE or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises an amino acid sequence as shown in any of the following GenBank accession numbers: AAA45779.1, AAA96680.1, ABI63526.1, ACM62297.1, ADD60055.1, ADD60132.1, ADM22391.1, ADM22468.1, ADM22544.1, ADM22621.1, ADM22698.1, ADM22775.1, ADM22851.1, ADM22928.1, ADM23005.1, ADM23081.1, ADM23157.1, ADM23233.1, A DM23311.1, ADM23385.1, ADM23459.1, ADM23533.1, ADM23607.1, ADM23682.1, ADM23757.1, ADM23833.1, ADN34689.1, ADN34692.1, ADN34695.1, AEQ77099.1, AER37649.1, AER37717.1, AER37788.1, AER37859.1, AER37931.1, AER38002.1, AER38072.1, AFA36179.1, AFA36180.1, AFA36181.1, AFA36182.1, AFA36183.1, AFA36184.1, AFA36185.1, AFA36186.1, AFA36187.1, AFA36188.1, AFA36189.1, AFA36190.1, AFA36191.1, AFA36192.1 , AFA36193.1, AFA36194.1, AFA36195.1, AFA36196.1, AFA36197.1, AFA36198.1, AFA36199.1, AFA36200.1, AFA36201.1, AFA36202.1, AFA36203.1 , AFE62896.1, AFI23659.1, AFK50417.1, AFP86432.1, AGZ01930.1, AIR95859.1, AJE60011.1, AJE60082.1, AJE60153.1, AJE60224.1, AJE60295. 1. AKE48647.1, AKE98373.1, AKE98374.1, AKE98375.1, AKE98376.1, AKE98377.1, AKE98378.1, AKE98379.1, AKE98380.1, AKE98381.1, AKE98382.1、AKE98383.1、AKE98384.1、AKE98385.1、AKE98386.1、AKE98387.1、AKE98 388.1、AKE98389.1、AKE98390.1、AKE98391.1、AKE98392.1、AKE98393.1、AK G59248.1, AKG59320.1, AKG59393.1, AKG59464.1, AKG59538.1, AKG59611.1, AKG59684.1, AKG59757.1, AKG59828.1, AKG59900.1, AKG59974.1, AKG600 48.1, AKG60120.1, AKG60191.1, AKG60263.1, AKG60336.1, AKG60406.1, AKG60476.1, AKG60548.1, AKG60622.1, AKG60694.1, AKG60765.1, AKG60837.1 、AKG60908.1、AKG60980.1、AKG61052.1、AKG61125.1、AKG61196.1、AKG612 69.1、AKG61341.1、AKG61413.1、AKG61486.1、AKG61558.1、AKG61631.1、AKG 61705.1, AKG61776.1, AKG61849.1, AKG61922.1, AKG61995.1, AKH80465.1, AKH80538.1, ALM22637.1, ALM22711.1, ALM22785.1, ALM22859.1, ALO1866 4.1, ALO18740.1, AMB65664.1, AMB65737.1, AMB65811.1, AMB65887.1, AMB65958.1, AMN09834.1, ANN83966.1, ANN84043.1, ANN84119.1, ANN84196.1 ANN84273.1、ANN84350.1、ANN84426.1、ANN84502.1、ANN84579.1、ANN8465 5.1、ANN84732.1、ANN84808.1、ANN84885.1、ANN84961.1、ANN85038.1、ANN8 5114.1, ANN85189.1, ANN85266.1, ANN85343.1, ANN85418.1, ANN85496.1, ANN85573.1, ANN85650.1, ANN85726.1, ANN85803.1, AOY34085.1, AOY36687.1. ARB08959.1, ARO38073.1, ARO38074.1, ARO38075.1, ARO38076.1, ARO38077.1, ARO38078.1, ARO3807 9.1, ARO38080.1, ASM47642.1, ASM47666.1, ASM47743.1, ASM47820.1, ASM47895.1, BAM73421.1, CAA26 062.1, CAA32272.1, CAF24756.1, CAF24757.1, CAF24758.1, CAF24759.1, CAF24760.1, CAF24761.1, CAF 24762.1, CAF24763.1, CAF24764.1, CAF24765.1, CAF24766.1, CAF24767.1, CAF24768.1, CAF24769.1, CA F24770.1, CAF24771.1, CAF24772.1, CAF24773.1, CAF24774.1, CAF24775.1, CAF24776.1, CAF24777.1, CAF24778.1, CAF24779.1, CAF24780.1, CAF24781.1, CAF24782.1, CAF24783.1, CAF24784.1, CAF24785.1 , P04290.1, P04488.1, P28986.1, Q703F0.1, SBO07910.1, SBS69571.1, SBS69576.1, SBS69595.1, SBS69 636.1, SBS69693.1, SBS69701.1, SBS69722.1, SBS69732.1, SBS69813.1, SBT69397.1 or YP_009137143.1. .

[0267] In some embodiments, the RNA encoding HSV gE as described herein comprises RNA encoding HSV-2 gE (also known as gE2) or an immunogenic fragment thereof.

[0268] In some embodiments, the nucleotide sequence of the RNA encoding the HSV-2 gE fragment comprises the following:

[0269]

[0270]

[0271] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent the 5' untranslated sequence (SEQ ID NO: 13). In some embodiments, bolded residues represent the signal sequence (leader sequence) (SEQ ID NO: 3). In some embodiments, italicized residues represent the 3' untranslated sequence (SEQ ID NO: 15) and the polyadenylated tail (SEQ ID NO: 16).

[0272] In another embodiment, the nucleotide sequence of the RNA encoding the HSV-2 gE fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof. In some embodiments, the sequence of the HSV-2 gE fragment is as shown in SEQ ID NO: 48.

[0273] In some embodiments, the HSV-2 gE fragment encoded by the RNA used in the methods and compositions of this disclosure comprises amino acids 24-405 from the gE of HSV-2 (e.g., strain 2.12 or US8), as shown in the following amino acid sequence:

[0274] RTSWKRVTSGEDVVLLPAPAGPEERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPVPTPTPDDYDEEDDAGVSERTPVSVPPPTPPRRPPVAPPTHPRVIPEVSH VRGVTVHMETPEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCAEMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRA (SEQ ID NO: 48).

[0275] In some embodiments, the HSV-2 gE fragment comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 48. In some embodiments, the HSV-2 gE fragment has the same amino acid sequence as SEQ ID NO: 48.

[0276] In some embodiments, the full-length HSV-2 gE encoded by the RNA used in the methods and compositions of this disclosure comprises the following amino acid sequence:

[0277] (SEQ ID NO: 49).

[0278] In some embodiments, HSV-2 gE comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence SEQ ID NO: 49. In some embodiments, HSV-2 gE has the same amino acid sequence as SEQ ID NO: 49.

[0279] In another embodiment, the HSV-2 gE or its immunogenic fragment encoded by the RNA used in the methods and compositions of this disclosure comprises an amino acid sequence as shown in any of the following GenBank accession numbers: ABU45436.1, ABU45437.1, ABU45438.1, ABU45439.1, ABW83306.1, ABW83308.1, ABW83310.1, ABW83312.1, ABW83314.1, ABW83316.1, ABW83318.1, ABW83320.1, ABW83322.1, ABW83324.1, ABW83326.1, ABW83328.1, ABW83330.1, ABW83332.1, A BW83334.1, ABW83336.1, ABW83338.1, ABW83340.1, ABW83342.1, ABW83344.1, ABW83346.1, ABW83348.1, ABW83350.1, ABW83352.1, ABW83354.1, ABW83356.1, ABW83358.1, ABW83360.1, ABW83362.1, ABW83364.1, ABW83366.1, ABW83368.1, ABW83370.1, ABW83372.1, ABW83374.1, ABW83376.1, ABW83378.1, ABW83380.1, ABW83382.1, ABW83384.1, ABW83386.1, ABW83388.1, ABW83390.1, ABW83392.1, ABW83394.1, ABW83396.1, ABW83398.1 , ABW83400.1, ABZ04069.1, AEV91407.1, AHG54732.1, AKC42830.1, AKC5 9307.1, AKC59378.1, AKC59449.1, AKC59520.1, AKC59591.1, AMB66104.1 , AMB66173.1, AMB66246.1, AMB66465.1, AQZ55756.1, AQZ55827.1, AQZ5 5898.1, AQZ55969.2, AQZ56040.2, AQZ56111.2, AQZ56182.1, AQZ56253. 2. AQZ56324.1, AQZ56395.1, AQZ56466.2, AQZ56537.1, AQZ56608.1, AQZ 56679.1, AQZ56750.1, AQZ56821.2, AQZ56892.1, AQZ56963.2, AQZ57034.2. AQZ57105.1, AQZ57176.1, AQZ57247.2, AQZ57318.2, AQZ57389.2, AQZ57460.2, AQZ57531.2, AQZ57602.2, AQZ57673.1, AQZ57744. 2. AQZ57815.1, AQZ57886.1, AQZ57957.2, AQZ58028.2, AQZ58099.1, AQZ58170.2, AQZ58241.2, AQZ58312.2, AQZ58383.2, AQZ58454.2 , AQZ58525.2, AQZ58596.1, AQZ58667.1, AQZ58738.2, AQZ58809.2, AQZ58880.2, AQZ58951.2, AQZ59022.2, AQZ59093.1, AQZ59164.1 , ARO38081.1, ARO38082.1, ARO38083.1, ARO38084.1, ARO38085.1, ARO38086.1, CAB06715.1, P89436.1, P89475.1 or YP_009137220.1. .

[0280] HSV glycoprotein H

[0281] In some embodiments, the compositions disclosed herein further comprise RNA or nucleoside-modified RNA encoding HSV glycoprotein H (gH) or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gH or an immunogenic fragment thereof comprises RNA encoding HSV-1 gH or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gH or an immunogenic fragment thereof comprises RNA encoding HSV-2 gH or an immunogenic fragment thereof.

[0282] In some embodiments, this document discloses a single composition comprising RNA encoding HSV gB and RNA encoding HSV gH or an immunogenic fragment thereof. In some embodiments, the gB and gH RNAs are portions of a single polynucleotide. In some embodiments, this document discloses two separate compositions to be administered as part of a single treatment, a first composition comprising RNA encoding HSV gB or an immunogenic fragment thereof, and a second composition comprising RNA encoding HSV gH or an immunogenic fragment thereof.

[0283] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, some uridine residues are 1-methyl-pseudouridine. In another embodiment, the nucleotide sequence of the RNA encoding the HSV-1 or HSV-2 gH fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof.

[0284] In some embodiments, the polynucleotide encoding an exemplary polypeptide comprising HSV-2 gH glycoprotein or an immunogenic fragment thereof comprises or is composed of SEQ ID NO: 189.

[0285] In other embodiments, the polynucleotide encoding an exemplary polypeptide comprising an HSV-2 gH glycoprotein or an immunogenic fragment thereof comprises or is composed of the following nucleotide sequences:

[0286]

[0287] In some embodiments, the polynucleotide encoding an exemplary polypeptide comprising gH glycoprotein or an immunogenic fragment thereof comprises greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 189 or SEQ ID NO: 189.

[0288] HSV glycoprotein L

[0289] In some embodiments, the compositions disclosed herein further comprise RNA or nucleoside-modified RNA encoding HSV glycoprotein L (gL) or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gL or an immunogenic fragment thereof comprises RNA encoding HSV-1 gL or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gL or an immunogenic fragment thereof comprises RNA encoding HSV-2 gL or an immunogenic fragment thereof.

[0290] In some embodiments, this document discloses a single composition comprising RNA encoding HSV gB and RNA encoding HSV gL or an immunogenic fragment thereof. In some embodiments, the gB and gL RNAs are portions of a single polynucleotide. In some embodiments, this document discloses two separate compositions to be administered as part of a single treatment, a first composition comprising RNA encoding HSV gB or an immunogenic fragment thereof, and a second composition comprising RNA encoding HSV gL or an immunogenic fragment thereof.

[0291] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, some uridine residues are 1-methyl-pseudouridine. In another embodiment, the nucleotide sequence of the RNA encoding the HSV-1 or HSV-2 gL fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof.

[0292] In some embodiments, the polynucleotide encoding an exemplary polypeptide comprising an HSV-2 gL glycoprotein or an immunogenic fragment thereof comprises or is composed of the following nucleotide sequences:

[0293] AGCATAAAAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTA TTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCGC TATGCGCATGCAGCTGCTGCTGCTGATCGCCCTGTCCCTGGCCCTGGTGACCAACTCCGGCTCCCAGGCCACCGAG TACGTGCTGCGCTCCGTGATCGCCAAGGAGGTGGGCGACATCCTGCGCGTGCCCTGCATGCGCACCCCCGCCGACG ACGTGTCCTGGCGCTACGAGGCCCCCTCCGTGATCGACTACGCCCGCATCGACGGCATCTTCCTGCGCTACCACTG CCCCGGCCTGGACACCTTCCTGTGGGACCGCCACGCCCAGCGCGCCTACCTGGTGAACCCCTTCCTGTTCGCCGCC GGCTTCCTGGAGGACCTGTCCCACTCCGTGTTCCCCGCCGACACCCAGGAGACCACCACCCGCCGCGCCCTGTACA AGGAGATCCGCGACGCCCTGGGCTCCCGCAAGCAGGCCGTGTCCCACGCCCCCGTGCGCGCCGGCTGCGTGAACTT CGACTACTCCCGCACCCGCCGCTGCGTGGGCCGCCGCGACCTGCGCCCCGCCAACACCACCTCCACCTGGGAGCCC CCCGTGTCCTCCGACGACGAGGCCTCCTCCCAGTCCAAGCCCCTGGCCACCCAGCCCCCCGTGCTGGCCCTGTCCA ACGCCCCCCCCCGCCGCGTGTCCCCCACCCGCGGCCGCCGCCGCCACACCCGCCTGCGCCGCAACtaataaactagtAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATA CCAACTTACACTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTT CACATTCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 19 0).

[0294] In other embodiments, the polynucleotide encoding an exemplary polypeptide comprising the HSV-2 gL glycoprotein or an immunogenic fragment thereof comprises or is composed of the following nucleotide sequences:

[0295] (SEQ ID NO: 191).

[0296] In some embodiments, the polynucleotide encoding an exemplary polypeptide comprising gL glycoprotein or an immunogenic fragment thereof comprises greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 191 or SEQ ID NO: 191.

[0297] HSV glycoprotein I

[0298] In some embodiments, the compositions disclosed herein further comprise RNA or nucleoside-modified RNA encoding HSV glycoprotein I (gI) or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gI or an immunogenic fragment thereof comprises RNA encoding HSV-1 gI or an immunogenic fragment thereof. In some embodiments, the RNA encoding HSV gI or an immunogenic fragment thereof comprises RNA encoding HSV-2 gI or an immunogenic fragment thereof.

[0299] In some embodiments, this document discloses a single composition comprising RNA encoding HSV gB and RNA encoding HSV gI or an immunogenic fragment thereof. In some embodiments, the gB and gI RNAs are portions of a single polynucleotide. In some embodiments, this document discloses two separate compositions to be administered as part of a single treatment, a first composition comprising RNA encoding HSV gB or an immunogenic fragment thereof, and a second composition comprising RNA encoding HSVGI or an immunogenic fragment thereof.

[0300] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, some uridine residues are 1-methyl-pseudouridine. In another embodiment, the nucleotide sequence of the RNA encoding the HSV-1 or HSV-2 gI fragment lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylated tail, or a combination thereof.

[0301] In some embodiments, the polynucleotide encoding an exemplary polypeptide comprising HSV-2 gI glycoprotein or an immunogenic fragment thereof comprises or is composed of SEQ ID NO: 184.

[0302] In some embodiments, this disclosure provides a polynucleotide encoding an HSV-2 gB glycoprotein or an immunogenic fragment thereof, or a truncated HSV-2 gI glycoprotein or an immunogenic fragment thereof. In some embodiments, this disclosure provides a polynucleotide encoding a full-length or untruncated HSV-2 gI glycoprotein. The HSV-2 gI glycoprotein is also referred to herein as gI2 and has all the same characteristics and limitations.

[0303] In some embodiments, the polynucleotides used in the methods, compositions and combinations of this disclosure encode the full-length HSV-2 gI glycoprotein comprising SEQ ID NO: 30.

[0304] In some embodiments, the polynucleotides used in the methods, compositions and combinations of this disclosure encode an HSV-2 gI immunogenic fragment comprising amino acids 25-230 of HSV-2 gI containing SEQ ID NO: 30.

[0305] In some embodiments, the HSV-2 gI glycoprotein or its immunogenic fragment or truncated HSV-2 gI glycoprotein or its immunogenic fragment comprises or consists of an amino acid sequence that is more than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 30.

[0306] In some embodiments, the polynucleotides used in the methods, compositions, and combinations of this disclosure further encode a signal sequence that encodes a signal peptide. In some embodiments, the signal peptide is an IL2 signal peptide (SEQ ID NO: 30).

[0307] In some embodiments, the modified RNA-encoded HSV-2 gI glycoprotein or its immunogenic fragments, or truncated HSV-2 gI glycoprotein or its immunogenic fragments, used in the methods, compositions, and combinations of this disclosure, comprises or consists of the amino acid sequence shown in any of the following GenBank accession numbers: ABW83313.1, ABW83327.1, ABW83385.1, ABW83397.1, AHG54731.1, AKC42829.1, AKC59306.1, AKC59377.1, AKC59519.1, AKC59590.1, AQZ56891.1, AQZ56962.2, AQZ58027.1, ATD86571.1, AT D86726.1, BAA00021.1, QAU10475.1, QAU10768.1, QBC74573.1, QBH76746.1, QBH76909.1, QBH78328.1, QBH78911.1, QBH80193.1, QBH80706.1, QBH80794.1, QBH81814.1, QBH82569.1, QBH83266.1, QBH84231.1, QBH85083.1, QBH85656.1 or SPT06174.1.

[0308] In some embodiments, the polynucleotides used in the methods, compositions, and combinations of this disclosure particularly encode an HSV-2 gI glycoprotein or an immunogenic fragment thereof comprising or truncated an HSV-2 gI glycoprotein or an immunogenic fragment thereof (e.g., amino acids 25-230):

[0309] GCCCAGGAGUCCUGGGCCGGCCCCACCGUGUCCCUGGUGUCCGACUCCCUGGUGGACGCCGGCGCCGUGGGCCCCCAGGGCUUCGUGGAGGAGGACCUGCGCGUGUUCGGCGAGCUGCACUUCGUGGGCGCCCAGGUGCCCCACACCAACUACUACGACGGCAUCAUCGAGCUGUUCCACUACCCCCUGGGCAACCACUGCCCCCGCGUGGUGCACGUGGUGACCCUGACCGCCUGCCCCCGCCGCCCCGCCGUGGCCUUCACCCUGUGCCGCUCCACCCACCACGCCCACUCCCCCGCCUACCCCACCCUGGAGCUGGGCCUGGCCCGCCAGCCCCUGCUGCGCGUGCGCACCGCCACCCGCGACUACGCCGGCCUGUACGUGCUGCGCGUGUGGGUGGGCUCCGCCACCAACGCCUCCCGCUUCGUGCUGGGCGUGGCCCUGUCCGCCAACGGCACCUUCGUGUACAACGGCUCCGACUACGGCUCCUGCGACCCCGCCCAGCUGCCCUUCUCCGCCCCCCGCCUGGGCCCCUCCUCCGUGUACACCCCCGGCGCCUCCCGCCCCACCCCCCCCCGCACCACCACCCCCCCCUCCUCCCCCCGCGACCCCACCCCCGCCCCCGGCGACACCGGCACCCCCGCCCCCGCCUCCGGCGAGAUCGCCCCCCCCAACUCCACCCGCUCCGCCUCCGAGUCCCGCCACCGCUAA (SEQ ID NO: 192)

[0310] In some embodiments, the nucleotide sequence encoding the HSV-2 gI glycoprotein or an immunogenic fragment thereof or a truncated HSV-2 gI glycoprotein or an immunogenic fragment thereof has a homology of greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 192.

[0311] In some embodiments, the polynucleotide encoding a polypeptide comprising HSV-2 gI glycoprotein or an immunogenic fragment thereof or a truncated HSV-2 gI glycoprotein or an immunogenic fragment thereof, as described herein, further comprises a signal sequence encoding a signal peptide. In other embodiments, the polynucleotide further comprises a non-protein-coding nucleotide sequence, such as a 5'UTR, 3'UTR, polyA tail, etc.

[0312] In some embodiments, the polynucleotides used in the methods, compositions, and combinations disclosed herein particularly comprise or consist of nucleotide sequences encoding an immunogenic fragment of the HSV-2 gI glycoprotein, signal sequences encoding a signal peptide, and non-protein-coding sequences (e.g., 5'UTR, 3'UTR, polyA tail, etc.).

[0313] In some embodiments, the polynucleotide comprises or consists of the following nucleotide sequences:

[0314]

[0315]

[0316] In some embodiments, the nucleotide sequence of the modified polynucleotide encoding the truncated HSV-2 gI contains greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 193.

[0317] In some embodiments, all uridine residues are 1-methyl-pseudouridine. In some embodiments, underlined residues represent a 5' untranslated sequence (SEQ ID NO: 193). In some embodiments, bolded residues represent a signal sequence (leader sequence) (SEQ ID NO: 193). In some embodiments, italicized residues represent a 3' untranslated sequence followed by a polyadenylated tail (SEQ ID NO: 193).

[0318] In some embodiments, the HSV gI glycoprotein encoded by a polynucleotide or an immunogenic fragment thereof, or a truncated HSV gI glycoprotein or an immunogenic fragment thereof, comprises or is composed of an HSV-1 gI domain involved in intercellular transmission. In another embodiment, the HSV gI glycoprotein encoded by a polynucleotide used in the methods, compositions, and combinations of this disclosure, or an immunogenic fragment thereof, or a truncated HSV gI glycoprotein or an immunogenic fragment thereof, comprises or is composed of an immune escape domain. In yet another embodiment, the HSV gI glycoprotein encoded by a polynucleotide used in the methods, compositions, and combinations of this disclosure, or an immunogenic fragment thereof, or a truncated HSV gI glycoprotein or an immunogenic fragment thereof, comprises a portion of or is composed of an immune escape domain.

[0319] In another embodiment, the HSV gI glycoprotein encoded by the polynucleotide used in the methods, compositions, and combinations of this disclosure, or an immunogenic fragment thereof or a truncated HSV gI glycoprotein or an immunogenic fragment thereof, has immunoprotective properties. In some embodiments, the protective immune response typically involves an antibody response. In another embodiment, mutants, sequence-conserved variants, and functionally conserved variants of the HSV gI glycoprotein or an immunogenic fragment thereof or a truncated HSV gI glycoprotein or an immunogenic fragment thereof may be used in the methods, compositions, and combinations of this disclosure, provided that all such variants retain the desired immunoprotective effect.

[0320] In some embodiments, the HSV gI glycoprotein or its immunogenic fragment, or a truncated HSV gI glycoprotein or its immunogenic fragment, may be derived from any strain of HSV. In some embodiments, the HSV gI glycoprotein or its immunogenic fragment, or a truncated HSV gI glycoprotein or its immunogenic fragment, may be derived from sequence variants of HSV, as seen in individuals infected with HSV.

[0321] In some embodiments, this disclosure provides RNA or nucleoside-modified RNA encoding a polypeptide comprising an extracellular domain of HSV-2 gB or an immunogenic fragment thereof, wherein the RNA further comprises a secretion signal sequence. In some embodiments, the secretion signal sequence encodes an IL2 secretion signaling polypeptide. In some embodiments, the IL2 secretion signaling polypeptide comprises or is composed of an amino acid sequence as shown in SEQ ID NO: 2. In another embodiment, the IL2 secretion signaling polypeptide is encoded by a ribonucleic acid sequence as shown in SEQ ID NO: 3.

[0322] In some embodiments, a single polynucleotide encodes an HSV gB glycoprotein or an immunogenic fragment thereof, as well as other HSV glycoproteins or immunogenic fragments thereof. In other embodiments, the HSV gB glycoprotein or an immunogenic fragment thereof, as well as other HSV glycoproteins or immunogenic fragments thereof, are encoded by a separate polynucleotide. In some embodiments, a single polynucleotide encodes a truncated HSV gB glycoprotein or an immunogenic fragment thereof, as well as other truncated HSV glycoproteins or immunogenic fragments thereof. In other embodiments, the truncated HSV gB glycoprotein or an immunogenic fragment thereof, as well as other truncated HSV glycoproteins or immunogenic fragments thereof, are encoded by a separate polynucleotide.

[0323] In some embodiments, the combinations disclosed herein comprise polynucleotides encoding immunogenic fragments of HSV-2 glycoprotein B (gB) antigen, HSV-2 glycoprotein C (gC) antigen, HSV-2 glycoprotein D (gD) antigen, and HSV-2 glycoprotein E (gE) antigen. In other embodiments, the combinations disclosed herein comprise polynucleotides encoding immunogenic fragments of HSV-2 gB antigen, HSV-2 gC antigen, HSV-2 gD antigen, HSV-2 gE antigen, and HSV-2 gI antigen.

[0324] In other embodiments, the combinations disclosed herein comprise polynucleotides encoding immunogenic fragments of HSV gB antigen, HSV gC antigen, and HSV gD antigen.

[0325] In some embodiments, the polynucleotides of this disclosure encode HSV polypeptides or fragments thereof that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequences listed in Table 1.

[0326] In some embodiments, the method of this disclosure comprises administering to a subject an HSV polypeptide or fragment thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence listed in Table 1.

[0327] Table 1: Exemplary amino acid sequences of HSV immunogens

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] In some embodiments, the polynucleotides or RNA disclosed herein comprise or consist of a ribonucleic acid sequence as shown in SEQ ID NO: 6, said ribonucleic acid sequence comprising a sequence encoding an IL2 signaling polypeptide and a truncated HSV-2 glycoprotein B.

[0339] In some embodiments, the polynucleotides of this disclosure comprise at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as those listed in Table 2.

[0340] In some embodiments, the method of this disclosure includes administering to a subject a polynucleotide comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same nucleotide sequence as those listed in Table 2.

[0341] Table 2: Exemplary nucleic acid sequences of HSV immunogens

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415] secretion signal

[0416] In some embodiments, the polynucleotides or RNA disclosed herein further comprise a secretion signal sequence encoding a secretion signaling polypeptide that functions, for example, in mammalian cells. In some embodiments, the secretion signal sequence encodes a modified secretion signaling polypeptide (e.g., containing amino acid substitutions or additions). In some embodiments, the secretion signal sequence is a codon-optimized secretion signal sequence.

[0417] In some embodiments, the secretory signal sequence used is a heterologous secretory signal sequence. In some embodiments, the heterologous secretory signal sequence comprises or is composed of a non-human secretory signal sequence. In some embodiments, the heterologous secretory signal sequence comprises or is composed of a viral secretory signal sequence. In some embodiments, the viral secretory signal sequence comprises or is composed of an HSV secretory signal sequence (e.g., an HSV-1 or HSV-2 secretory signal sequence). In some embodiments, the secretory signal sequence comprises or is composed of an HSV-1 secretory signal sequence. In some embodiments, the secretory signal sequence comprises or is composed of an HSV-2 secretory signal sequence. In some embodiments, the secretory signal sequence encodes a secretory signal polypeptide characterized by a length of about 15 to 30 amino acids. In some embodiments, the secretory signal sequence encodes a secretory signal polypeptide that preferably allows the transport of HSV-1 glycoprotein or its immunogenic fragment, HSV-2 glycoprotein or its immunogenic fragment, or both, and said secretory signal polypeptide associates with a defined cellular compartment, preferably on the cell surface, in the endoplasmic reticulum (ER), or in an endosome-lysosome compartment.

[0418] In some embodiments, the secretion signal sequence is the natural secretion signal sequence of the encoded glycoprotein.

[0419] Suitable secretion signal sequences are generally known in the art; for example, the signal sequence of human tissue plasminogen activator (tPA). Generally, the signal sequence encodes a signal peptide, a short peptide located at the N-terminus of a protein, carrying information about protein secretion. In some embodiments, the signal peptide may be derived from prokaryotes or eukaryotes. In some embodiments, the signal peptide contains 25-30 residues. In other embodiments, longer signal peptides, such as eukaryotic signal sequences, may be used. In some embodiments, the signal peptide contains up to about 140 residues. In some embodiments, the secretion signal sequence in the polynucleotide described herein is an HSV secretion signal.

[0420] In some embodiments, the secretion signal sequence encodes an IL2 secretion signal peptide. In some embodiments, the IL2 secretion signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 2. In another embodiment, the IL2 secretion signal peptide is encoded by a ribonucleic acid sequence shown in SEQ ID NO: 3. In another embodiment, the secretion signal sequence encodes an HSV gB secretion signal peptide. In some embodiments, the HSV gB secretion signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 4. In another embodiment, the secretion signal sequence encodes an HSV gD secretion signal peptide, and in one embodiment, the secretion signal peptide is an HSV-2 gD secretion signal peptide. In another embodiment, the secretion signal sequence encodes an HSV protein secretion signal peptide, and in one embodiment, the secretion signal peptide comprises an HSV glycoprotein secretion signal. In one embodiment, the HSV glycoprotein secretion signal is an HSV-2 glycoprotein secretion signal.

[0421] In some embodiments, this disclosure provides a polynucleotide encoding a polypeptide comprising an extracellular domain of HSV-2 gB or an immunogenic fragment thereof, wherein the RNA further comprises a secretion signal sequence encoding an HSV gB secretion signaling polypeptide. In some embodiments, the HSV gB secretion signaling polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 4.

[0422] In some embodiments, the RNA disclosed herein comprises a secretion signal sequence encoding a secretion signal peptide listed in Table 3 or a secretion signal peptide differing therefrom by one, two, three, four, or five amino acids. In some embodiments, the secretion signal peptide is selected from the secretion signal peptides listed in Table 3 and is functionally linked to the N-terminus of an HSV immunogen selected from those listed in Table 1.

[0423] Table 3: Exemplary signal peptides

[0424]

[0425]

[0426] In some embodiments, the secretion signal sequence comprises a sequence listed in Table 4 or a signal sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence listed in Table 4. In some embodiments, the secretion signal sequence is selected from the secretion signal sequences listed in Table 4 and is functionally linked (i.e., within the box) to the 5' end of an HSV immunogen nucleic acid sequence selected from those listed in Table 2.

[0427] Table 4: Exemplary Signal Sequences

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434] Exemplary nucleotide sequence features

[0435] In some embodiments, the polynucleotides or RNA disclosed herein further include a 5' untranslated region. In some embodiments, the polynucleotides or RNA disclosed herein further include a 3' untranslated region. In some embodiments, the nucleotide sequences described herein may include a 5' cap, which may be integrated during transcription or conjugated to the nucleotide sequence post-transcriptionally.

[0436] In some embodiments, the polynucleotides or RNA disclosed herein further include a polyA tail. In some embodiments, this disclosure provides an RNA construct comprising (i) a 5' untranslated region, (ii) a polynucleotide as disclosed herein, (iii) a 3' untranslated region, and (iv) a polyA tail sequence. In some embodiments, the 5' untranslated region is derived from tobacco etch virus. In some embodiments, the 3' untranslated region is derived from Xenopus β-globulin.

[0437] In some embodiments, the polynucleotides or RNA disclosed herein further comprise a cap-independent translation enhancer. In some embodiments, the cap-independent translation enhancer is a tobacco etch virus (TEV) cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is any other cap-independent translation enhancer known in the art. Each possibility represents a separate embodiment of this disclosure.

[0438] In other embodiments, the polynucleotides of the methods, compositions, and combinations of this disclosure further comprise internal ribosome entry sites (IRES). In some embodiments, the IRES comprises a 5' leader IRES and a cistron IRES in the 1.8 kb family of Immediate Early Transcription (IRES) 1; foot-and-mouth disease virus IRES; cricket paralysis virus IRES; hepatitis A IRES; hepatitis C IRES; Kaposi's sarcoma-associated herpesvirus IRES; plague virus IRES; microRNA virus IRES; cereal aphid virus IRES; or combinations thereof.

[0439] In other embodiments, the polynucleotides of the methods, compositions, and combinations of this disclosure further comprise a cap-independent translation enhancer (CITE). In another embodiment, the polynucleotides of the methods, compositions, and combinations of this disclosure do not comprise a CITE. In some embodiments, the CITE is a tobacco etch virus (TEV) cap-independent translation enhancer. In other embodiments, the CITE comprises a TED (translation-enhancing domain); a BTE (BYDV-like translational element); a PTE (PMV-like translational element); a TSS (T-shaped structure); a Y-shaped structure; an I-shaped structure; or a combination thereof. In other embodiments, the cap-independent translation enhancer is any other cap-independent translation enhancer known in the art. Each possibility represents a separate embodiment of this disclosure.

[0440] In some embodiments, the polynucleotides or RNA disclosed herein further comprise an m7GpppG cap, a 3'-O-methyl-m7GpppG cap, or an anti-reverse cap analogue.

[0441] In some embodiments, the polynucleotides or RNA disclosed herein further comprise one or more non-protein-coding sequences. In some embodiments, the non-protein-coding sequences perform one or more of the following: increase protein expression, regulate innate and adaptive immunogenicity, improve intracellular delivery and localization. In some embodiments, the non-protein-coding sequences relate to precursor mRNA processing, RNA stability, and translation initiation. Non-protein-coding sequences having the above functions are generally known in the art.

[0442] In some embodiments, the nucleotide sequences provided herein encode one or more glycoproteins (e.g., HSV gB) or immunogenic fragments thereof. In some embodiments, the polynucleotide comprises a 5' cap, a 5' UTR, a nucleotide sequence encoding one or more glycoproteins (e.g., HSV gB) or immunogenic fragments thereof, a 3' UTR, and a polyA tail.

[0443] 1.5' cap

[0444] The structural feature of natural messenger RNA (mRNA) is a cap structure at the five-terminal (5') end. Natural eukaryotic mRNA contains a 7-methylguanosine cap linked to the mRNA via a 5'-to-5'-triphosphate bridge, forming the cap0 structure (m7GpppN). Additional modifications can occur at the 2'-hydroxyl (2'-OH) position of the first and subsequent nucleotides (e.g., the 2'-hydroxyl may be methylated to form 2'-O-Me), producing the "cap1" and "cap2" five-terminal ends, respectively. By citing Diamond et al., (2014) *Cytokine & Growth Factor Reviews*, 25:543–550, which are incorporated herein by reference in their entirety, translation of cap0-RNA is less efficient than that of cap1-RNA, with the 2'-O-Me at the penultimate position of the 5' end of the RNA playing a decisive role. The absence of 2'-O-met has been shown to trigger innate immunity and activate interferon (IFN) responses. Daffis et al. (2010) Nature, 468:452-456; and Züst et al. (2011) Nature Immunology, 12:137-143, each of which is incorporated herein by reference in its entirety.

[0445] RNA capping has been well studied and described, for example, in Decroly E et al. (2012) Nature Reviews 10: 51–65; and Ramanathan A. et al. (2016) Nucleic Acids Research; 44(16): 7511–7526, the entire contents of each of which are incorporated herein by reference. For example, in some embodiments, the 5'-cap structure applicable to the context of this invention is cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA ("anti-reverse cap analog"), modified ARCA (e.g., ARCA modified with thiophosphate), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deazono-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0446] As used herein, the term "5' cap" refers to a structure present at the 5' end of RNA (e.g., mRNA) and typically comprises a guanosine nucleotide (also known as Gppp or G(5')ppp(5')) linked to RNA (e.g., mRNA) via a 5' to 5' triphosphate bond. In some embodiments, the guanosine nucleotide included in the 5' cap may be modified, for example by methylation at one or more sites (e.g., at the 7 site) on the base (guanine) and / or by methylation at one or more sites on the ribose. In some embodiments, the guanosine nucleotide included in the 5' cap comprises 3'O methylation at the ribose (3'OMeG). In some embodiments, the guanosine nucleotide included in the 5' cap comprises methylation at the 7 site of guanine (m7G). In some embodiments, the guanosine nucleotide included in the 5' cap comprises methylation at the 7 site of guanine and 3'O methylation at the ribose (m7(3'OMeG)). It should be understood that the identifiers used in the above paragraphs, such as “(m27,3'-O)G” or “m7(3'OMeG)”, are applicable to other structures described herein.

[0447] In some embodiments, providing RNA with the 5'-cap disclosed herein can be achieved through in vitro transcription, wherein the 5'-cap is co-transcribed into the RNA strand, or it can be attached to the RNA post-transcriptionally using a capping enzyme. In some embodiments, co-transcriptional capping with the cap disclosed herein improves the capping efficiency of RNA compared to co-transcriptional capping with a suitable reference. In some embodiments, improved capping efficiency can increase RNA translation efficiency and / or translation rate, and / or increase the expression of the encoded protein. In some embodiments, alteration of the polynucleotide generates a non-hydrolyzable cap structure, which can, for example, prevent decapping and increase the RNA half-life.

[0448] In some embodiments, the 5' cap used is a cap0, cap1, or cap2 structure. See, for example, Ramanathan A et al. Figure 1 and Decroly E et al. Figure 1 Each of the aforementioned documents is incorporated herein by reference in its entirety. In some embodiments, the RNA described herein comprises a cap1 structure. In some embodiments, the RNA described herein comprises a cap2 structure.

[0449] In some embodiments, the RNA described herein comprises a cap0 structure. In some embodiments, the cap0 structure comprises a methylated guanosine nucleotide at the 7-position of guanine ((m7)G). In some embodiments, such a cap0 structure is linked to the RNA via a 5' to 5' triphosphate bond and is also referred to herein as (m7)Gppp. In some embodiments, the cap0 structure comprises a methylated guanosine nucleotide at the 2' position of the ribose of guanosine. In some embodiments, the cap0 structure comprises a methylated guanosine nucleotide at the 3' position of the ribose of guanosine. In some embodiments, the guanosine nucleotide included in the 5' cap comprises methylation at the 7-position of guanine and at the 2' position of the ribose ((m27,2'-O)G). In some embodiments, the guanosine nucleotide included in the 5' cap comprises methylation at the 7-position of guanine and at the 2' position of the ribose ((m27,3'-O)G).

[0450] In some embodiments, the cap1 structure comprises a guanosine nucleoside methylated at the 7' position of guanine and optionally methylated at the 2' or 3' position of ribose ((m7)G), and a first nucleotide in RNA methylated at the 2'O position ((m2'-O)N1). In some embodiments, the cap1 structure comprises a guanosine nucleoside methylated at the 7' position of guanine and the 3' position of ribose ((m7)G), and a first nucleotide in RNA methylated at the 2'O position ((m2'-O)N1). In some embodiments, the cap1 structure is linked to RNA via a 5' to 5' triphosphate bond and is also referred herein, for example, as ((m7)Gppp(2'-O)N1) or ((m27,3'-O)Gppp(2'-O)N1), where N1 is as defined and described herein. In some embodiments, the cap1 structure includes a second nucleotide N2 located at position 2 and selected from A, G, C or U, such as (m7)Gppp(2'-O)N1pN2 or (m27,3'-O)Gppp(2'-O)N1pN2, wherein each of N1 and N2 is as defined and described herein.

[0451] In some embodiments, the cap2 structure comprises a guanosine nucleoside methylated at the 7' position of guanine and optionally methylated at the 2' or 3' position of ribose ((m7)G), and a first and second nucleotide with 2'O methylation in RNA ((m2'-O)N1p(m2'-O)N2). In some embodiments, the cap2 structure comprises a guanosine nucleoside methylated at the 7' position of guanine and the 3' position of ribose ((m7)G), and a first and second nucleotide with 2'O methylation in RNA. In some embodiments, the cap2 structure is linked to RNA via 5' to 5' triphosphate bonds and is also referred herein, for example, as ((m7)Gppp(2'-O)N1p(2'-O)N2) or ((m27,3'-O)Gppp(2'-O)N1p(2'-O)N2), wherein each of N1 and N2 is as defined and described herein.

[0452] In some embodiments, the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure containing N1, wherein N1 is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G*N1, wherein N1 is as defined above and herein, and G* contains the structure of formula (I):

[0453]

[0454] (I)

[0455] or its salt,

[0456] Each of R2 and R3 is -OH or -OCH3; and X is O or S.

[0457] In some embodiments, R2 is -OH. In some embodiments, R2 is -OCH3. In some embodiments, R3 is -OH. In some embodiments, R3 is -OCH3. In some embodiments, R2 is -OH and R3 is -OH. In some embodiments, R2 is -OH and R3 is -CH3. In some embodiments, R2 is -CH3 and R3 is -OH. In some embodiments, R2 is -CH3 and R3 is -CH3.

[0458] In some embodiments, X is O. In some embodiments, X is S.

[0459] In some embodiments, the 5' cap is a dinucleotide cap0 structure (e.g., (m7)GpppN1, (m27,2'-O)GpppN1, (m27,3'-O)GpppN1, (m7)GppSpN1, (m27,2'-O)GppSpN1, or (m27,3'-O)GppSpN1), wherein N1 is G. In some embodiments, the 5' cap is a dinucleotide cap0 structure (e.g., (m7)GpppN1, (m27,2'-O)GpppN1, (m27,3'-O)GpppN1, (m7)GppSpN1, (m27,2'-O)GppSpN1, or (m27,3'-O)GppSpN1), where N1 is A, U, or C. In some embodiments, the 5' cap is a dinucleotide cap1 structure (e.g., (m7)Gppp(m2'-O)N1, (m27,2'-O)Gppp(m2'-O)N1, (m27,3'-O)Gppp(m2'-O)N1, (m7)GppSp(m2'-O)N1, (m27,2'-O)GppSp(m2'-O)N1 or (m27,3'-O)GppSp(m2'-O)N1), wherein N1 is as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of: (m7)GpppG (“Ecap0”), (m7)Gppp(m2'-O)G (“Ecap1”), (m27,3'-O)GpppG (“ARCA” or “D1”), and (m27,2'-O)GppSpG (“β-S-ARCA”). In some embodiments, the 5' cap is (m7)GpppG (“Ecap0”) having the structure of formula (II):

[0460]

[0461] (II)

[0462] Or its salt.

[0463] In some embodiments, the 5' cap is (m7)Gppp(m2'-O)G (“Ecap1”), having the structure of formula (III):

[0464]

[0465] (III)

[0466] Or its salt.

[0467] In some embodiments, the 5' cap is (m27,3'-O)GpppG (“ARCA” or “D1”), having the structure of formula (IV):

[0468]

[0469] (IV)

[0470] Or its salt.

[0471] In some embodiments, the 5' cap is (m27,2'-O)GppSpG (“β-S-ARCA”), having the structure of formula (V):

[0472]

[0473] (V)

[0474] Or its salt.

[0475] In some embodiments, the 5' cap is a trinucleotide cap structure. In some embodiments, the 5' cap is a trinucleotide cap structure comprising N1pN2, wherein N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G*N1pN2, wherein N1 and N2 are as defined above and herein, and G* comprises the structure of formula (VI):

[0476]

[0477] (VI)

[0478] or its salt, wherein R 2 R 3 X is as defined and described in this article.

[0479] In some embodiments, the 5' cap is a trinucleotide cap0 structure (e.g., (m7)GpppN1pN2, (m27,2'-O)GpppN1pN2, or (m27,3'-O)GpppN1pN2), wherein N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide cap1 structure (e.g., (m7)Gppp(m2'-O)N1pN2, (m27,2'-O)Gppp(m2'-O)N1pN2, (m27,3'-O)Gppp(m2'-O)N1pN2), wherein N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide cap2 structure (e.g., (m7)Gppp(m2'-O)N1p(m2'-O)N2, (m27,2'-O)Gppp(m2'-O)N1p(m2'-O)N2, (m27,3'-O)Gppp(m2'-O)N1p(m2'-O)N2), wherein N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of: (m27,3'-O)Gppp(m2'-O)ApG ("CleanCap AG", "CC413"), (m27,3'-O)Gppp(m2'-O)GpG ("CleanCap GG"), (m7)Gppp(m2'-O)ApG, (m7)Gppp(m2'-O)GpG, (m27,3'-O)Gppp(m26,2'-O)ApG and (m7)Gppp(m2'-O)ApU.

[0480] In some embodiments, the 5' cap is (m27,3'-O)Gppp(m2'-O)ApG ("CleanCap AG", "CC413"), having the structure of formula (VII):

[0481]

[0482] (VII)

[0483] Or its salt.

[0484] In some embodiments, the 5' cap is (m27,3'-O)Gppp(m2'-O)GpG ("CleanCap GG"), having the structure of formula (VIII):

[0485]

[0486] (VIII)

[0487] Or its salt.

[0488] In some embodiments, the 5' cap is (m7)Gppp(m2'-O)ApG, having the structure of formula (IX):

[0489]

[0490] (IX)

[0491] Or its salt.

[0492] In some embodiments, the 5' cap is (m7)Gppp(m2'-O)GpG, having the structure of formula (X):

[0493]

[0494] (X)

[0495] Or its salt.

[0496] In some embodiments, the 5' cap is (m27,3'-O)Gppp(m26,2'-O)ApG, having the structure of formula (XI):

[0497]

[0498] (XI)

[0499] Or its salt.

[0500] In some embodiments, the 5' cap is (m7)Gppp(m2'-O)ApU, having the structure of formula (XII):

[0501]

[0502] (XII)

[0503] Or its salt.

[0504] In some embodiments, the 5' cap is a tetranucleotide cap structure. In some embodiments, the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap G*N1pN2pN3, wherein N1, N2, and N3 are as defined above and herein, and G* comprises the structure of formula (XIII):

[0505]

[0506] (XIII)

[0507] or its salt, wherein R 2 R 3 X is as defined and described in this article.

[0508] In some embodiments, the 5' cap is a tetranucleotide cap0 structure (e.g., (m 7 )GpppN1pN2pN3、(m2 7,2'-O )GpppN1pN2pN3 or (m2 7,3'-O )GpppN1N2p N3), where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide Cap1 structure (e.g., (m 7 )Gppp(m 2'-O )N1pN2pN3、(m2 7,2'-O ) Gppp(m 2'-O )N1pN2pN3、(m2 7,3'-O )Gppp(m 2'-O )N1pN2N3), where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide Cap2 structure (e.g., (m 7 )Gppp(m 2'-O )N1p(m 2'-O )N2pN3、(m2 7,2'-O )Gppp(m 2'-O )N1p (m 2'-O )N2pN3、(m2 7,3'-O )Gppp(m 2'-O )N1p(m 2'-O )N2pN3), where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of: (m2 7,3'-O )Gppp(m 2'-O Ap(m) 2'-O )GpG、(m2 7 ,3'-O )Gppp(m 2'-O )Gp(m 2'-O GpC, (m 7 )Gppp(m 2'-O Ap(m) 2'-O UpA and (m 7 )Gppp(m 2'-O Ap(m) 2’-O )GpG.

[0509] In some embodiments, the 5' cap is (m2) 7,3'-O )Gppp(m 2'-O Ap(m) 2'-O GpG has the structure of formula (XIV):

[0510]

[0511] (XIV)

[0512] Or its salt.

[0513] In some embodiments, the 5' cap is (m2) 7,3'-O )Gppp(m 2'-O )Gp(m 2'-O GpC has the structure of formula (XV):

[0514]

[0515] (XV)

[0516] Or its salt.

[0517] In some embodiments, the 5' cap is (m 7 )Gppp(m 2'-O Ap(m) 2'-O UpA has the structure of formula (XVI):

[0518]

[0519] (XVI)

[0520] Or its salt.

[0521] In some embodiments, the 5' cap is (m 7 )Gppp(m 2'-O Ap(m) 2'-O GpG has the structure of formula (XVII):

[0522]

[0523] (XVII)

[0524] Or its salt.

[0525] 2. Proximal sequence of the cap

[0526] In some embodiments, the 5' UTR used according to this disclosure includes, for example, a cap proximal sequence as disclosed herein. In some embodiments, the cap proximal sequence includes a sequence adjacent to the 5' cap. In some embodiments, the cap proximal sequence includes nucleotides at positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0527] In some embodiments, the cap structure comprises one or more polynucleotides in a proximal cap sequence. In some embodiments, the cap structure comprises m 7 The guanosine cap and the nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure includes m 7 The guanosine cap and the nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure includes m 7The guanosine cap and the nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises m 7 The guanosine cap and the nucleotides of the RNA polynucleotide are +1, +2, and +3 (N1, N2, and N3).

[0528] Those skilled in the art will understand upon reading this disclosure that, in some embodiments, one or more residues of the cap proximal sequence (e.g., one or more residues of residues +1, +2, +3, +4, and / or +5) can be included in the RNA by being incorporated into the cap entity (e.g., a cap1 or cap2 structure, etc.); alternatively, in some embodiments, at least some residues of the cap proximal sequence can be added enzymatically (e.g., by a polymerase, such as T7 polymerase). For example, m2 is used therein. 7,3'-O Gppp(m1 2'-O In some exemplary embodiments of the ApG cap, (m1) is the cap. 2'-O The A and G residues. The +1 (i.e., N1) and +2 (i.e., N2) and +3, +4 and +5 are added by polymerase (e.g. T7 polymerase).

[0529] In some embodiments, the 5'' cap is a dinucleotide cap structure, wherein the proximal sequence of the cap contains N1 of the 5' cap, where N1 is any nucleotide, such as A, C, G, or U. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the proximal sequence of the cap contains N1 and N2 of the 5' cap, where N1 and N2 are independently any nucleotide, such as A, C, G, or U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., the tetranucleotide cap structures described above and herein), wherein the proximal sequence of the cap contains N1, N2, and N3 of the 5' cap, where N1, N2, and N3 are any nucleotides, such as A, C, G, or U.

[0530] In some embodiments, for example, where the 5' cap is a dinucleotide cap structure, the proximal sequence of the cap includes N1 and N2, N3, N4, and N5 of the 5' cap, wherein N1 to N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some embodiments, for example, where the 5' cap is a trinucleotide cap structure, the proximal sequence of the cap includes N1 and N2 and N3, N4, and N5 of the 5' cap, wherein N1 to N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some embodiments, for example, where the 5' cap is a tetranucleotide cap structure, the proximal sequence of the cap includes N1, N2, and N3 and N4 and N5 of the 5' cap, wherein N1 to N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0531] In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N5 is C. In some embodiments, N5 is G. In some embodiments, N5 is U. It should be understood that each embodiment (e.g., for N1 to N5) in the embodiments described above and herein can be used alone or in combination, and / or can be combined with other embodiments of the variables (e.g., 5' cap) described above and herein.

[0532] In some embodiments, the proximal cap sequence comprises A1 and G2 of the Cap1 structure, and includes the sequence A3A4U5 at positions +3, +4, and +5 of the nucleotide sequence, respectively.

[0533] 3.5' UTR

[0534] In some embodiments, the RNA used according to this disclosure comprises a 5' UTR. In some embodiments, the 5'-UTR may comprise multiple different sequence elements; in some embodiments, such diversity may be or comprise multiple copies of one or more specific sequence elements (e.g., possibly from a specific source or otherwise referred to as functional or characteristic sequence elements). In some embodiments, the 5' UTR comprises multiple different sequence elements.

[0535] The term “untranslated region” or “UTR” is generally used in the art to refer to a region in a DNA molecule that has been transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA polynucleotide. An untranslated region (UTR) can be located at the 5' end (upstream) of an open reading frame (5' UTR) and / or the 3' end (downstream) of an open reading frame (3' UTR). As used herein, the term “5' UTR” refers to the sequence of nucleotides between the 5' end of a nucleotide sequence (e.g., a transcription start site) and the start codon of the coding region of the nucleotide sequence. In some embodiments, “5' UTR” refers to a sequence of nucleotides that begins at the 5' end of a nucleotide sequence (e.g., a transcription start site) and ends one nucleotide (nt) before the start codon (typically AUG) of the coding region of the nucleotide sequence, for example, in its natural background. In some embodiments, the 5' UTR contains a Kozak sequence. The 5' UTR is located downstream of the 5' cap (if present), for example, directly adjacent to the 5' cap. In some embodiments, the 5' UTR disclosed herein includes a cap proximal sequence, such as those defined and described herein. In some embodiments, the cap proximal sequence includes a sequence adjacent to the 5' cap.

[0536] Exemplary 5' UTRs include human alpha globulin (hAg) 5' UTR or a fragment thereof, TEV 5' UTR or a fragment thereof, HSP70 5' UTR or a fragment thereof, or c-Jun 5' UTR or a fragment thereof.

[0537] In some embodiments, the RNA disclosed herein comprises hAg 5' UTR or a fragment thereof.

[0538] In some embodiments, the RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a 5' UTR having the sequence GGAATAAAAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGC (SEQ ID NO: 13). In some embodiments, the RNA disclosed herein comprises a 5' UTR having the sequence shown in SEQ ID NO: 13.

[0539] In some embodiments, the RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a 5' UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 194). In some embodiments, the RNA disclosed herein comprises a 5' UTR having the sequence shown in SEQ ID NO: 194.

[0540] In some embodiments, the RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a 5' UTR having the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 195), corresponding to the hAg-Kozak 5' UTR. In some embodiments, the RNA disclosed herein comprises a 5' UTR having the sequence shown in SEQ ID NO: 195.

[0541] 4. PolyA tail

[0542] In some embodiments, the polynucleotides (e.g., DNA, RNA) disclosed herein comprise, for example, a polyadenosine monophosphate (polyA) sequence as described herein. In some embodiments, the polyA sequence is located downstream of the 3' UTR, for example, adjacent to the 3' UTR.

[0543] As used herein, the term "poly(A) sequence" or "polyA tail" refers to a sequence of continuous or interrupted adenosine residues typically located at the 3' end of an RNA polynucleotide. Poly(A) sequences are known to those skilled in the art and may follow the 3' UTR in the RNA described herein. A continuous poly(A) sequence is characterized by a continuous sequence of adenosine residues. Continuous poly(A) sequences are typical in nature. In some embodiments, the polynucleotides disclosed herein comprise continuous poly(A) sequences. In some embodiments, the polynucleotides disclosed herein comprise interrupted poly(A) sequences. In some embodiments, the RNA disclosed herein may have a poly(A) sequence that is attached to the free 3' end of the RNA post-transcriptionally by a template-dependent RNA polymerase or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0544] It has been demonstrated that the poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells as well as on protein levels translated from an open reading frame located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017, which is incorporated herein by reference).

[0545] In some embodiments, the poly(A) sequence according to this disclosure is not limited to a specific length; in some embodiments, the poly(A) sequence has any length. In some embodiments, the poly(A) sequence comprises at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 A nucleotides, and specifically about 120 A nucleotides, substantially composed of or composed of. In this context, "substantially composed of" means that, based on the number of nucleotides in the poly(A) sequence, the majority of the nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "composed of" means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

[0546] In some embodiments, a poly(A) sequence is attached during RNA transcription, for example, during the preparation of in vitro transcribed RNA based on a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as a poly(A) box.

[0547] In some embodiments, the poly(A) box present in the coding strand of DNA is generally composed of dA nucleotides but interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides. Such boxes are disclosed in WO 2016 / 005324 A1, which is hereby incorporated by reference. Any poly(A) box disclosed in WO 2016 / 005324 A1 may be used according to this disclosure, which is incorporated herein by reference in its entirety. Poly(A) boxes, generally composed of dA nucleotides but interrupted by equally distributed random sequences of the four nucleotides (dA, dC, dG, and dT) and having a length of, for example, 5 to 50 nucleotides, are shown to cover the constant proliferation of plasmid DNA in *E. coli* at the DNA level and remain associated with beneficial properties supporting RNA stability and translation efficiency at the RNA level. In some embodiments, the poly(A) sequence contained in the RNA polynucleotide described herein is generally composed of A nucleotides but is interrupted by random sequences of four nucleotides (A, C, G, and T). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides.

[0548] In some embodiments, no nucleotides other than A are side-attached to the 3' end of the poly(A) sequence, meaning that the poly(A) sequence is not masked or followed by nucleotides other than A at its 3' end.

[0549] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly(A) sequence may consist substantially of at least 20, at least 30, at least 40, at least 80, or at least 100, and at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0550] In some embodiments, the poly(A) sequence contains a specific number of adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, about 150, or about 200. In some embodiments, the poly(A) sequence of RNA may contain 200 or fewer A residues. In some embodiments, the poly(A) sequence of RNA may contain about 200 A residues. In some embodiments, the poly(A) sequence of RNA may contain 180 or fewer A residues. In some embodiments, the poly(A) sequence of RNA may contain about 180 or fewer A residues. In some embodiments, the poly(A) sequence may contain 150 or fewer residues.

[0551] In some embodiments, the RNA comprises a poly(A) sequence comprising the following nucleotide sequence: AAA ...

[0552] In some embodiments, the RNA comprises a poly(A) sequence containing the following nucleotide sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCA UAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 196) or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 196. In some embodiments, the poly(A) sequence comprises a plurality of A residues interrupted by a linker. In some embodiments, the linker comprises the nucleotide sequence GCAUAUGAC.

[0553] 5.3' UTR

[0554] In some embodiments, the RNA used according to this disclosure comprises a 3' UTR. As used herein, the terms "tri-start untranslated region," "3' untranslated region," or "3' UTR" refer to the sequence of an RNA molecule that begins after a stop codon in the coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon in the coding region of an open reading frame sequence, for example, in its natural environment. In other embodiments, the 3' UTR does not begin immediately after a stop codon in the coding region of an open reading frame sequence, for example, in its natural environment. In some embodiments, the term "3' UTR" preferably does not include a poly(A) sequence. Thus, the 3' UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0555] In some embodiments, the RNA disclosed herein comprises a 3' TR including an F element and / or an I element. In some embodiments, the 3' UTR or its proximal sequence contains a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an XhoI site.

[0556] In some embodiments, the RNA construct includes an F element. In some embodiments, the F element sequence is the 3' UTR of an N-terminal cleavage enhancer (AES).

[0557] In some embodiments, the RNA disclosed herein comprises a 3' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence having CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 186). In some embodiments, the RNA disclosed herein comprises a 3' UTR having the sequence having SEQ ID NO: 186.

[0558] In some embodiments, the RNA disclosed herein comprises a 3' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence having CTAGTAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCT (SEQ ID NO: 198). In some embodiments, the RNA disclosed herein comprises a 3' UTR having the sequence shown in SEQ ID NO: 198.

[0559] In some embodiments, 3' UTR is a FI element as described in WO2017 / 060314, which is incorporated herein by reference in its entirety.

[0560] In some embodiments, the polynucleotide as described herein comprises a 5' untranslated region, a 3' untranslated region, a polyA tail, a 5' cap, or a combination thereof.

[0561] In some embodiments, the nucleotide sequences disclosed herein are optimized according to techniques known in the art. For example, codon optimization is a genetic engineering method to improve gene expression by altering synonymous codons according to an organism's codon preferences. As is well known in the art, nucleotide alterations can be made in the whole nucleotide sequence of interest according to an organism's codon usage preferences to improve translation efficiency and thereby increase protein expression without altering the protein sequence.

[0562] Nucleoside modification

[0563] In some embodiments, this disclosure provides compositions comprising modified RNA and methods of using them. In some embodiments, the modified RNA comprises one or more modified nucleoside residues. For example, in some embodiments, RNA comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the nucleotide sequence listed in Table 2 or Table 4 comprises one or more modified nucleoside residues.

[0564] In some embodiments, RNA as described herein refers to messenger RNA.

[0565] In some embodiments, all uridine residues are modified as described herein. In some embodiments, one or more of the RNAs described herein are nucleoside-modified RNAs. In other embodiments, two or more of the RNAs described herein are nucleoside-modified RNAs. In other embodiments, three or more of the RNAs described herein are nucleoside-modified RNAs.

[0566] In some embodiments, the modified nucleoside of the methods and compositions of this disclosure is m5C (5-methylcytidine). In another embodiment, the modified nucleoside is m5U (5-methyluridine). In another embodiment, the modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0567] In some embodiments, the modified nucleoside is m 1 A (1-methyladenosine), m 2 A (2-methyladenosine), m 6 A (N6-methyladenosine), Am (2'-O-methyladenosine), ms 2 m 6 A (2-methylthio-N6-methyladenosine), i 6 A (N6-Isopentenyladenosine), ms 2 i 6 A (2-methylthio-N6-isopentenyl adenosine), io 6 A (N6-(cis-hydroxyisopentenyl)adenosine), ms 2 io 6 A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g 6 A (N6-glycine carbamoyl adenosine), t 6 A (N6-threonylcarbamoyladenosine), ms 2 t 6 A (2-methylthio-N6-threonylcarbamoyladenosine), m 6 t 6 A (N6-methyl-N6-threonylcarbamoyladenosine), hn 6 A (N6-hydroxyn-valinecarbamoyladenosine), ms 2 hn 6 A (2-methylthio-N6-hydroxyvaline carbamoyl adenosine), Ar(p) (2'-O-ribosyl adenosine (phosphate)), I (inosine), m 1I (1-methylinosine), m 1 Im (1,2'-O-dimethylinosine), m 3 C(3-methylcytidine), m 5 C(5-methylcytidine), Cm(2'-O-methylcytidine), s 2 C(2-thiocytidine), ac 4 C (N4-acetylcytidine), f 5 C(5-formylcytidine), m 5 Cm(5,2'-O-dimethylcytidine), ac 4 Cm (N4-acetyl-2'-O-methylcytidine), k 2 C (lysicin), m 1 G (1-methylguanosine), m 2 G (N2-methylguanosine), m 7 G(7-methylguanosine), Gm(2'-O-methylguanosine), m 2 2G (N2,N2-dimethylguanosine), m 2 Gm(N2,2'-O-dimethylguanosine), m 2 2Gm (N2,N2,2'-O-trimethylguanosine), Gr(p) (2'-O-ribosylguanosine (phosphate)), yW (huaitoside), o2yW (peroxyhuaitoside), OHyW (hydroxyhuaitoside), OHyW* (undermodified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxy resin pigmentoside), galQ (galactosyl-pigmentoside), manQ (mannoside pigmentoside), preQ0 (7-cyano-7-deazonoguanosine), preQ1 (7-aminomethyl-7-deazonoguanosine), G + (Archaetoside), Ψ (Pseudouridine), D (Dihydrouridine), m 5 U (5-methyluridine), Um (2'-O-methyluridine), m 5 Um (5,2'-O-dimethyluridine), m 1 Ψ(1-methylpseuuridine), Ψm(2'-O-methylpseuuridine), s 2 U (2-thiouridine), s 4 U (4-thiouridine), m 5 s 2 U (5-methyl-2-thiouridine), s 2 Um (2-thio-2'-O-methyluridine), ACP 3 U (3-(3-amino-3-carboxypropyl)uridine), ho 5 U (5-hydroxyuridine), mo 5 U (5-methoxyuridine), cmo5 U (uridine 5-oxyacetic acid), mcmo 5 U (uridine 5-oxyacetic acid methyl ester), chm 5 U (5-(carboxyhydroxymethyl)uridine), mchm 5 U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm 5 U (5-methoxycarbonylmethyluridine), mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm 5 s 2 U (5-methoxycarbonylmethyl-2-thiouridine), nm 5 s 2 U (5-aminomethyl-2-thiouridine), mnm 5 U (5-methylaminomethyluridine), mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine), mnm 5 se 2 U (5-methylaminomethyl-2-selenouridine), ncm 5 U (5-carbamoylmethyluridine), ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm 5 U (5-Carboxymethylaminomethyluridine), cmnm 5 Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm 5 s 2 U (5-carboxymethylaminomethyl-2-thiouridine), m 6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m 4 C(N4-methylcytidine), m 4 Cm(N4,2'-O-dimethylcytidine), hm 5 C(5-hydroxymethylcytidine), m 3 U (3-methyluridine), m 1 acp 3 Ψ (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine), cm 5 U (5-carboxymethyluridine), m 6 Am(N6,2'-O-dimethyladenosine), m 6 2Am(N6,N6,2'-O-trimethyladenosine), m 2,7 G(N2,7-dimethylguanosine), m 2,2,7 G(N2,N2,7-trimethylguanosine), m 3Um (3,2'-O-dimethyluridine), m 5 D (5-methyldihydrouridine), m 3 Ψ (3-methylpseuuridine), f 5 Cm (5-formyl-2'-O-methylcytidine), m 1 Gm (1,2'-O-dimethylguanosine), m 1 Am(1,2'-O-dimethyladenosine), τm 5 U (5-tauronic acid methyluridine), τm 5 s 2 U (5-tauronic acid methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), ac 6 A (N6-acetyladenosine), inm 5 U (5-(isopentenylaminomethyl)uridine), inm 5 s 2 U (5-(isopentenylaminomethyl)-2-thiouridine), inm 5 Um (5-(isopentenylaminomethyl)-2'-O-methyluridine), m 2,7 Gm (N2,7,2'-O-trimethylguanosine), m 4 2Cm(N4,N4,2'-O-trimethylcytidine), C + (guanidine), m 8 A (8-methyladenosine), gmnm 5 s 2 U (geranylated 5-methylaminomethyl-2-thiouridine), gcmnm 5 s 2 U (geranylated 5-carboxymethylaminomethyl-2-thiouridine) or CMN 5 U (5-cyanomethyluridine).

[0568] In some embodiments, the modified nucleoside residues are pseudouridine or pseudouridine family residues.

[0569] In some embodiments, the modified RNA contains pseudouridine residues. In some embodiments, pseudouridine refers to the C-glycosidic isomer of the nucleoside uridine. In some embodiments, the pseudouridine residues contain m 1 acp 3 Ψ (1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine), m 1 Ψ(1-methylpseuuridine), Ψm(2'-O-methylpseuuridine), m 5 D (5-methyldihydrouridine), m 3Ψ (3-methylpseudouridine) or combinations thereof. In some embodiments, the pseudouridine residue comprises a 1-methylpseudouridine residue, rather than uridine.

[0570] In some embodiments, the modified nucleoside residues are pseudouridine analogs. In some embodiments, "pseudouridine analog" is any modification, variant, subtype, or derivative of pseudouridine. For example, pseudouridine analogs include, but are not limited to, 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-tauronicomethyl-pseudouridine, 1-tauronicomethyl-4-thio-pseudouridine, and 1-methylpseudouridine (m 1 Ψ), 1-methyl-4-thio-pseuuridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazona-pseudouridine, 2-thio-1-methyl-1-deazona-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp) 3 Ψ) and 2'-O-methyl-pseuuridine (Ψm).

[0571] In some embodiments, the modified nucleobase is modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (Ψ), pyridin-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, and 2-thio-uridine (s). 2 U), 4-thiouridine (s) 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho) 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxyuridine (mo) 5 U), uridine 5-oxyacetic acid (cmo) 5 U), uridine 5-oxyacetic acid methyl ester (cmo) 5 U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm) 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm) 5 U), 5-methoxycarbonylmethyl-uridine (mcm) 5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm) 5 s 2 U), 5-methylaminomethyluridine (mnm) 5 U), 5-methylaminomethyl-2-thio-uridine (mnm) 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm) 5 se 2 U), 5-carbamoylmethyluridine (ncm) 5 U), 5-Carboxymethylaminomethyluridine (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uridine (cmnm) 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauronic acid methyl-uridine (τcm) 5 U), 1-tauronic acid methyl-pseuuridine, 5-tauronic acid methyl-2-thio-uridine (τrm) 5 s 2 U), 1-tauronic acid methyl-4-thio-pseuuridine, 5-methyl-uridine (m 5 U, i.e., deoxythymidine with nucleobases), 1-methylpseuuridine (m 1 Ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-pseuuridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazona-pseudouridine, 2-thio-1-methyl-1-deazona-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m) 5 D), 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as 1-methylpseudouridine (m 1 Ψ), 3-(3-amino-3-carboxypropyl)uridine (acp) 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp) 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm) 5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thiouridine, 2'O-methyluridine (Um), 5,2'-O-dimethyluridine (m) 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyluridine (s) 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2'-β-methyluridine (ncm) 5 Um), 5-Carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm) 5 Um), 3,2'-O-dimethyluridine (m) 3 Um), 5-(isopentenylaminomethyl)-2'-β-methyluridine (inm) 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-arasu-uridine, 2'-F-uridine, 2'-OH-arasu-uridine, 5-(2-carbonylmethoxyvinyl)uridine and 5-[3-(1-E-propenylamino)uridine].

[0572] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudo-isocytidine, and 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac) 4 C), 5-formyl-cytidine (f) 5 C), N4-methyl-cytidine (m) 4 C), 5-methyl-cytidine (m) 5 C) 5-Halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm) 5 C), 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thio-cytidine (s 2 C), 2-Thio-5-methyl-cytidine, 4-Thio-pseudo-cytidine, 4-Thio-1-methyl-pseudo-cytidine, 4-Thio-1-methyl-1-deaza-pseudo-cytidine, 1-methyl-1-deaza-pseudo-cytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m)5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac) 4 Cm), N4,2'-O-dimethyl-cytidine (m) 4 Cm), 5-formyl-2'-O-methyl-cytidine (f) 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m) 4 2Cm), 1-thio-cytidine, 2'-F-arasyl-cytidine, 2'-F-cytidine and 2'-OH-arasyl-cytidine.

[0573] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deazido-adenosine, 7-deazido-8-azido-adenosine, 7-deazido-2-amino-purine, 7-deazido-8-azido-2-amino-purine, 7-deazido-2,6-diamino-purine, 7-deazido-8-azido-2,6-diamino-purine, and 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m) 6 A) 2-Methylthio-N6-methyl-adenosine (ms) 2 m 6 A), N6-isopentenyl-adenosine (i 6 A) 2-Methylthio-N6-isopentenyl-adenosine (ms) 2 i 6 A) N6-(cis-hydroxyisopentenyl)adenosine (io 6 A) 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms) 2 io 6 A), N6-glycylcarbamoyl-adenosine (g) 6 A), N6-threonylcarbamoyl-adenosine (t) 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m) 6 t 6 A) 2-Methylthio-N6-threonylcarbamoyl-adenosine (ms) 2 g 6 A), N6,N6-dimethyl-adenosine (m) 6 2A), N6-hydroxyn-valine amide carbamoyl-adenosine (hn) 6A), 2-Methylthio-N6-hydroxyn-valine acylcarbamoyl-adenosine (ms) 2 hn 6 A) N6-acetyl-adenosine (ac) 6A ), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenine, 2'-O-methyl-adenine (Am), N6,2'-O-dimethyl-adenine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m) 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Am), 2'-β-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arasyl-adenosine, 2'-F-adenosine, 2'-OH-arasyl-adenosine and N6-(19-amino-pentaenodecyl)-adenosine.

[0574] In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (M... 1 I), Wyorogosin (imG), Methylwyorogosin (mimG), 4-Demethyl-Wyorogosin (imG-14), Isoorogosin (imG2), Wyorogosin (yW), Peroxywayorogosin (o2yW), Hydroxywayorogosin (OHyW), Inadequately Modified Hydroxywayorogosin (OHyW*), 7-Deazono-Guanosin, Brassin (Q), Epoxy Resin Brassin (oQ), Galactosyl-Guanosin (galQ), Mannosyl-Guanosin (manQ), 7-Cyano-7-Deazono-Guanosin (preQ0), 7-Aminomethyl-7-Deazono-Guanosin (preQ1), Archaeoside (G) + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m) 2 G), N2,N2-dimethyl-guanosine (m) 2 2G), N2,7-dimethyl-guanosine (m 2 ,7G), N2,N2,7-dimethyl-guanosine (m 2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m) 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m) 2'7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m) 1 Im) and 2'-O-ribosylguanosine (phosphate) (Gr(p)).

[0575] The nucleobases of a nucleotide can be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. For example, each nucleobase can be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase may also include, for example, naturally occurring and synthetic base derivatives, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated (e.g., 8-bromo), 8-amino, 8-thiol, 8-thiouracil, etc. Alkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (specifically 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; and 1,3,5-triazine. When nucleotides are represented by the abbreviations A, G, C, T, or U, each letter refers to the corresponding base and / or its derivative. For example, A includes adenine or adenine analogues (e.g., 7-deazoadenine).

[0576] Modifications on nucleoside interbonds

[0577] Modified nucleotides, which can be incorporated into polynucleotides, major constructs, or RNA molecules, can be modified at nucleoside internucleotides (e.g., phosphate backbones). In this document, the phrases “phosphate ester” and “phosphate diester” are used interchangeably in the context of a polynucleotide backbone. The backbone phosphate ester group can be modified by replacing one or more oxygen atoms with different substituents. Further, modified nucleosides and nucleotides can include large-scale replacement of the unmodified phosphate ester portion with another nucleoside internucleotide as described herein. Examples of modified phosphate ester groups include, but are not limited to, thiophosphates, selenophosphates, boranophosphates, boranophosphateesters, hydrogen phosphates, aminophosphates, diaminophosphates, alkyl or aryl phosphonates, and triphosphates. In dithiophosphates, both non-linked oxygen atoms are replaced with sulfur. The phosphate ester linker can also be modified by replacing the linking oxygen with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylene-phosphonate).

[0578] The α-thiosubstituted phosphate moiety imparts stability to RNA and DNA polymers via non-natural thiophosphate backbone bonds. Thiophosphate DNA and RNA exhibit increased nuclease resistance and thus longer half-lives in the cellular environment. Thiophosphate-linked polynucleotides, major constructs, or modified RNA molecules are also expected to reduce innate immune responses through weaker binding / activation of cellular innate immune molecules.

[0579] In a particular embodiment, the modified nucleoside includes α-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseuuridine).

[0580] Other nucleoside bonds that may be used according to this disclosure are described below, including nucleoside bonds that do not contain phosphorus atoms.

[0581] A combination of modified sugars, nucleobases, and nucleoside bonds.

[0582] The polynucleotides, major constructs, and modified RNAs disclosed herein may include combinations of modifications to sugars, nucleobases, and / or nucleoside bonds.

[0583] In another embodiment, the purification formulation of RNA, oligonucleotides, or polynucleotides of the methods and compositions of this disclosure comprises a combination of two or more of the above modifications. In another embodiment, the purification formulation of RNA or oligonucleotides comprises a combination of three or more of the above modifications. In yet another embodiment, the purification formulation of RNA or oligonucleotides comprises a combination of more than three of the above modifications.

[0584] In some embodiments, the modified RNA comprises modified RNA synthesized in vitro.

[0585] In some embodiments, this disclosure includes one or more modified RNAs encoding HSV glycoproteins. In some embodiments, the modified RNA contains pseudouridine or pseudouridine family residues. In another embodiment, the modified RNA of this disclosure is capable of directing protein expression of the HSV glycoprotein encoded thereon.

[0586] In another embodiment, this disclosure provides an in vitro transcribed RNA molecule encoding an HSV glycoprotein containing pseudouridine. In yet another embodiment, this disclosure provides a synthetic RNA molecule encoding an HSV glycoprotein containing pseudouridine.

[0587] In another embodiment, the in vitro transcribed RNA molecule of the methods and compositions of this disclosure is synthesized by T7 phage RNA polymerase. In another embodiment, the molecule is synthesized by SP6 phage RNA polymerase. In another embodiment, the molecule is synthesized by T3 phage RNA polymerase. In another embodiment, the molecule is synthesized by a polymerase selected from the above polymerases. In another embodiment, the RNA is chemically synthesized on a column similar to that used for DNA.

[0588] In another embodiment, the nucleoside modified in the RNA, oligonucleotide, or polynucleotide of the methods and compositions of this disclosure is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenine (A). In another embodiment, the modified nucleoside is guanine (G).

[0589] In another embodiment, the RNA of the methods and compositions of this disclosure further comprises a polyA tail. In another embodiment, the RNA of the methods and compositions of this disclosure does not comprise a polyA tail. Each possibility represents a separate embodiment of this disclosure.

[0590] In another embodiment, the RNA of the methods and compositions of this disclosure comprises an m7GpppG cap. In another embodiment, the RNA of the methods and compositions of this disclosure does not comprise an m7GpppG cap. In another embodiment, the RNA of the methods and compositions of this disclosure does not comprise 3'-O-methyl-m7GpppG. In another embodiment, the RNA of the methods and compositions of this disclosure comprises an irreversible cap analog, which, in some embodiments, is added during RNA transcription. In another embodiment, the RNA of the methods and compositions of this disclosure comprises an anti-reverse cap analog. Each possibility represents a separate embodiment of this disclosure.

[0591] In another embodiment, the RNA of the methods and compositions of this disclosure further comprises a cap-independent translation enhancer. In another embodiment, the RNA of the methods and compositions of this disclosure does not contain a cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is a tobacco etch virus (TEV) cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is any other cap-independent translation enhancer known in the art. Each possibility represents a separate embodiment of this disclosure.

[0592] In some embodiments, "pseudouridine" refers to m 1 acp 3 Ψ (1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine). In another embodiment, the term refers to m 1 Ψ(1-methylpseudouridine). In another embodiment, the term refers to Ψm(2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 D (5-methyldihydrouridine). In another embodiment, the term refers to m 3 Ψ(3-methylpseuuridine). In another embodiment, the modified nucleoside is 4'(pseuuridine). In another embodiment, the term refers to the unmodified pseudouridine moiety. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the pseudouridines described above. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of this disclosure.

[0593] In another embodiment, the modified RNA comprises a modified nucleoside, and in some embodiments, the modified nucleoside comprises m 5 C, m5U, m 6 A, s 2 U, Ψ, 2'-O-methyl-U, 2'-O-methyl pseudouridine or combinations thereof.

[0594] In another embodiment, this disclosure provides a method for delivering a recombinant protein to a subject, the method comprising the step of contacting the subject with RNA of the method and composition of this disclosure, thereby delivering the recombinant protein to the subject.

[0595] In another embodiment, the method of this disclosure includes increasing the number, percentage, or frequency of modified uridine nucleotides in an RNA molecule to reduce immunogenicity or improve translation efficiency. In some embodiments, the number of modified uridine residues in the RNA, oligonucleotide, or polynucleotide molecule determines the magnitude of the effect observed in this disclosure.

[0596] In another embodiment, 0.1% to 100% of the uridine residues in the modified RNA of the methods and compositions of this disclosure are modified (e.g., by the presence of pseudouridine). In another embodiment, 0.1% of the residues are modified. In another embodiment, 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the score is 25%. In another embodiment, the score is 30%. In another embodiment, the score is 35%. In another embodiment, the score is 40%. In another embodiment, the score is 45%. In another embodiment, the score is 50%. In another embodiment, the score is 60%. In another embodiment, the score is 70%. In another embodiment, the score is 80%. In another embodiment, the score is 90%. In another embodiment, the score is 100%.

[0597] In another embodiment, the score is less than 5%. In another embodiment, the score is less than 3%. In another embodiment, the score is less than 1%. In another embodiment, the score is less than 2%. In another embodiment, the score is less than 4%. In another embodiment, the score is less than 6%. In another embodiment, the score is less than 8%. In another embodiment, the score is less than 10%. In another embodiment, the score is less than 12%. In another embodiment, the score is less than 15%. In another embodiment, the score is less than 20%. In another embodiment, the score is less than 30%. In another embodiment, the score is less than 40%. In another embodiment, the score is less than 50%. In another embodiment, the score is less than 60%. In another embodiment, the score is less than 70%.

[0598] In another embodiment, 0.1% of the residues of a given uridine nucleotide are modified. In another embodiment, the fraction of nucleotides is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the score is 45%. In another embodiment, the score is 50%. In another embodiment, the score is 60%. In another embodiment, the score is 70%. In another embodiment, the score is 80%. In another embodiment, the score is 90%. In another embodiment, the score is 100%.

[0599] In another embodiment, the fraction of a given uridine nucleotide is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0600] In another embodiment, the terms "ribonucleotide," "oligonucleotide," and "polynucleotide" refer in some embodiments to compounds comprising nucleotides in which the sugar portion is ribose. In another embodiment, the term includes both RNA and RNA derivatives in which the backbone is modified. Various RNA backbone modifications are known in the art and are contemplated in this disclosure. In some embodiments, the modified RNA is PNA (peptide nucleic acid). PNA contains a peptide backbone and nucleotide bases and is, in another embodiment, capable of binding to both DNA and RNA molecules. In another embodiment, the nucleotide is modified by replacing one or more phosphodiester bonds with thiophosphate bonds. In another embodiment, the artificial nucleic acid comprises any other variant of the phosphate backbone of a natural nucleic acid known in the art. Each nucleic acid derivative represents a separate embodiment of this disclosure.

[0601] Methods for generating nucleic acids having a modified backbone are well known in the art and described, for example, in U.S. Patents 5,723,335 and 5,663,153, Hutcherson et al., and related PCT Publication WO95 / 26204. Each method represents a separate embodiment of this disclosure.

[0602] The nucleic acid of interest can be purified by any method known in the art or any method to be developed, provided that the purification method removes impurities from the nucleic acid preparation and thereby significantly reduces the immunogenicity potential of the nucleic acid preparation. In some embodiments, the nucleic acid of interest is purified using high-performance liquid chromatography (HPLC). In another embodiment, the nucleic acid of interest is purified by contacting the nucleic acid of interest with the bacterial enzyme RNase III. In various other embodiments, any method of nucleic acid purification that substantially reduces the immunogenicity of the nucleic acid preparation can be used. Non-limiting examples of purification methods that can be used with the compositions and methods of this disclosure include liquid chromatography separation and enzymatic digestion, each method used alone or in any combination, simultaneously or in any order. Non-limiting examples of liquid chromatography separation include HPLC and rapid protein liquid chromatography (FPLC). Useful materials in the HPLC and FPLC methods of this disclosure include, but are not limited to, cross-linked polystyrene / divinylbenzene (PS / DVB), PS / DVB-C18, alkylated PS / DVB, helical DNA columns (Varian), Eclipse dsDNA analytical columns (Agilent Technologies), reversed-phase 5 (RPC-5) exchange material, DNAPac, ProSwift, and bioinert UltiMate.RTM. 3000 titanium columns (Dionex). Useful enzymes in the enzymatic digestion methods of this disclosure include any enzyme capable of digesting any contaminants in the nucleic acid formulations of this disclosure, such as dsRNA contaminants, and including, but not limited to, RNase III, RNase V1, Dicer, and Chipper (see Fruscoloni et al., 2002, Proceedings of the National Academy of Sciences (PNAS) 100:1639). Non-limiting examples of assays for assessing the purity of the nucleic acid of interest include dot hybridization assays, northern blot assays, and dendritic cell activation assays, as described elsewhere herein.

[0603] In another embodiment, the immunogenicity of the modified RNA of the methods and compositions of this disclosure is significantly lower than that of an unmodified in vitro synthesized RNA molecule having the same sequence. In another embodiment, the immunogenicity of the modified RNA molecule is half that of its unmodified counterpart. In another embodiment, the immunogenicity is reduced by 3-fold. In another embodiment, the immunogenicity is reduced by 5-fold. In another embodiment, the immunogenicity is reduced by 7-fold. In another embodiment, the immunogenicity is reduced by 10-fold. In another embodiment, the immunogenicity is reduced by 15-fold. In another embodiment, the immunogenicity is reduced by half. In another embodiment, the immunogenicity is reduced by 50-fold. In another embodiment, the immunogenicity is reduced by 100-fold. In another embodiment, the immunogenicity is reduced by 200-fold. In another embodiment, the immunogenicity is reduced by 500-fold. In another embodiment, the immunogenicity is reduced by 1000-fold. In another embodiment, the immunogenicity is reduced by 2000-fold. In another embodiment, the immunogenicity is reduced by another factor.

[0604] In another embodiment, "significantly reduced immunogenicity" refers to a detectable reduction in immunogenicity. In another embodiment, the term refers to a fold decrease in immunogenicity (e.g., 1 in the fold decreases enumerated above). In another embodiment, the term refers to a reduction such that an effective amount of modified RNA can be administered without inducing a detectable immune response. In another embodiment, the term refers to a reduction such that repeated administration of modified RNA can be performed without inducing an immune response sufficient to produce detectable reduced expression of the recombinant protein. In another embodiment, the reduction allows repeated administration of modified RNA without inducing an immune response sufficient to eliminate detectable expression of the recombinant protein.

[0605] Methods for determining immunogenicity are well known in the art and are described in detail in U.S. Patent 8,278,036, which is hereby incorporated by reference.

[0606] In another embodiment, the modified RNA of the methods and compositions of this disclosure is translated more efficiently in cells compared to unmodified RNA molecules having the same sequence. In another embodiment, the modified RNA exhibits enhanced translation ability by target cells. In another embodiment, translation is enhanced by 2-fold compared to its unmodified counterpart. In another embodiment, translation is enhanced by 3-fold. In another embodiment, translation is enhanced by 5-fold. In another embodiment, translation is enhanced by 7-fold. In another embodiment, translation is enhanced by 10-fold. In another embodiment, translation is enhanced by 15-fold. In another embodiment, translation is enhanced by 20-fold. In another embodiment, translation is enhanced by 50-fold. In another embodiment, translation is enhanced by 100-fold. In another embodiment, translation is enhanced by 200-fold. In another embodiment, translation is enhanced by 500-fold. In another embodiment, translation is enhanced by 1000-fold. In another embodiment, translation is enhanced by 2000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200 times. In another embodiment, the factor is 10-300 times. In another embodiment, the factor is 10-500 times. In another embodiment, the factor is 20-1000 times. In another embodiment, the factor is 30-1000 times. In another embodiment, the factor is 50-1000 times. In another embodiment, the factor is 100-1000 times. In another embodiment, the factor is 200-1000 times. In another embodiment, the translation enhances any other significant amount or range of amounts. Each possibility represents a separate embodiment of this disclosure.

[0607] Methods for determining translation efficiency are well known in the art and include, for example, measuring the activity of the encoded reporter protein (e.g., luciferase or renilla or green fluorescent protein [Wall AA, Phillips AM et al., Effective translation of the second cistron in two Drosophila dicistronic transcripts is determined by the absence of in-frame AUG codons in the first cistron. Journal of Biochemistry 2005; 280(30): 27670-8]), or measuring the radioactive label incorporated into the translated protein (Ngosuwan J, Wang NM et al., Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of pre-secretory proteins into the endoplasmic reticulum). (Reticulum). Journal of Biochemistry 2003; 278(9): 7034-42). Each method represents a separate embodiment of this disclosure.

[0608] Codon optimization and GC enrichment

[0609] This disclosure also provides a codon-optimized nucleotide sequence.

[0610] As used herein, the term "codon optimization" refers to altering codons in the coding region of a nucleic acid molecule (e.g., a nucleotide sequence) to reflect typical codon usage in a host organism (e.g., a subject receiving the nucleic acid molecule (e.g., a nucleotide sequence), preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, in some embodiments, the coding region is codon-optimized using the RNA molecule described herein for optimal expression in a subject to treatment. In some embodiments, codon optimization may be performed such that a codon that yields high-frequency tRNA is inserted in place of a "rare codon." In some embodiments, codon optimization may include increasing the G / C content of the coding region of the RNA described herein, relative to the guanosine / cytosine (G / C) content of the corresponding coding sequence of wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably unmodified compared to the amino acid sequence.

[0611] In some embodiments, the coding sequence (also referred to as the “coding region”) is codon-optimized to express the composition (e.g., a pharmaceutical composition) in a subject (e.g., a human) to which it is administered. Therefore, in some embodiments, the sequence (e.g., the nucleotide sequence) in such a polynucleotide may differ from the wild-type sequence encoding the associated antigen, fragment thereof, or epitope, even if the amino acid sequence of said antigen, fragment thereof, or epitope is wild-type.

[0612] In some embodiments, a codon optimization strategy for expression in relevant subjects (e.g., humans) and even, in some cases, in specific cells or tissues.

[0613] Different species exhibit specific preferences for certain codons of particular amino acids. Without expecting to be bound by any single theory, codon preference (differences in codon use between organisms) is generally associated with RNA translation efficiency, which is further considered to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of a chosen tRNA in a cell can broadly reflect the most frequently used codons in peptide synthesis. Therefore, genes can be tailored based on codon optimization to optimize gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" at www.kazusa.orjp / codon / , and these tables can be modified in various ways. Computer algorithms for codon optimization of specific sequences for expression in specific subjects or their cells are also available, such as GeneForge (Aptagen; Jacobus, PA).

[0614] In some embodiments, the polynucleotides of this disclosure (e.g., polynucleotides or nucleotide sequences) are codon-optimized, wherein codon adjustments in the polynucleotide (e.g., polynucleotides) are for human codon use (referred to herein as "human codon-optimized polynucleotides"). In some embodiments, a portion of the nucleotide sequence is codon-optimized (e.g., a portion or portion encoding a glycoprotein or a portion or portion encoding a signal sequence). In some embodiments, the entire nucleotide sequence is codon-optimized. Codons encoding the same amino acid occur at different frequencies in subjects, such as humans. Therefore, in some embodiments, the coding sequence of the polynucleotides of this disclosure is modified such that the frequency of codons encoding the same amino acid corresponds to the frequency of the codons naturally present according to human codon use, for example as shown in Table 5. For example, in the case of amino acid Ala, the wild-type coding sequence is preferably adjusted such that codon "GCC" is used at a frequency of 0.40, codon "GCT" at a frequency of 0.28, codon "GCA" at a frequency of 0.22, and codon "GCG" at a frequency of 0.10, etc. (see Table 5). Therefore, in some embodiments, such procedures (as exemplified with Ala) are applied to each amino acid encoded by the coding sequence of a polynucleotide to obtain a sequence adapted for use with human codons.

[0615] Table 5: Human codon usage with indications of the frequency of each amino acid.

[0616]

[0617]

[0618] Certain strategies for codon optimization and / or G / C enrichment for human expression have been described in WO2002 / 098443, which is incorporated herein by reference in its entirety. In some embodiments, coding sequences may be optimized using multi-parameter optimization strategies. In some embodiments, optimization parameters may include parameters that affect protein expression, such as those that may be affected at the transcriptional, RNA, and / or translational levels. In some embodiments, exemplary optimization parameters include, but are not limited to: transcriptional level parameters (including, for example, GC content, consensus splicing sites, recessive splicing sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); RNA level parameters (including, for example, unstable RNA motifs, ribosome entry sites, repetitive sequences, and combinations thereof); translational level parameters (including, for example, codon usage, premature Poly(A) sites, ribosome entry sites, secondary structures, and combinations thereof); or combinations thereof.In some embodiments, the coding sequence can be optimized using algorithms such as the GeneOptimizer algorithm, as described in Fath et al., “Multiparameter RNA and CodonOptimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression,” PLoS ONE 6(3): e17596; Rabb et al., “The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization,” Systems and Synthetic Biology (2010) 4:215-225, which are incorporated herein by reference in their entirety; and Graft et al., “Codon-optimized genes that enable increased heterologous expression in mammalian cells and induce a robust immune response in mice after vaccination with naked DNA.” "Expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA" (Methods Mol Med, 2004) 94:197-210, the entire contents of each of these references are incorporated herein by reference for the purposes described herein. In some embodiments, the coding sequence may be optimized using the Eurofins tuning and optimization algorithm "GENEius," as described in the Eurofins application notes: Eurofins tuning and optimization software "GENEius," compared to other optimization algorithms, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0619] In some embodiments, the G / C content of the coding sequences used according to this disclosure is increased compared to the coding sequences of the HSV gC, gD, and / or gE (or their immunogenic fragments) constructs described herein. In some embodiments, the guanosine / cytidine (G / C) content of the coding regions is modified relative to the equivalent coding sequences of the HSV gC, gD, and / or gE (or their immunogenic fragments) constructs described herein, but the amino acid sequences encoded by the nucleotide sequences are not modified.

[0620] Without being bound by any particular theory, GC enrichment is believed to improve the translation of payload sequences. Generally, sequences with increased G (guanosine) / C (cytidine) content are more stable than sequences with increased A (adenosine) / U (uridine) content. Given the fact that several codons encode the same amino acid (so-called genetic code degeneracy), the codons most favorable for stability (so-called alternative codon use) can be determined. Depending on the amino acid to be encoded by the nucleotide sequence, there are several possibilities for modification of the ribonucleic acid sequence compared to its wild-type sequence. Specifically, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U, or contain less A and / or U nucleotides.

[0621] In some embodiments, the G / C content of the coding region of the nucleotide sequence described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G / C content of the coding region before codon optimization of, for example, wild-type RNA. In some embodiments, the G / C content of the coding region of the nucleotide sequence described herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G / C content of the coding region before codon optimization of, for example, wild-type RNA.

[0622] In some embodiments, the stability and translation efficiency of the nucleotide sequence may be incorporated into one or more elements that are confirmed to contribute to the stability and / or translation efficiency of the nucleotide sequence; exemplary such elements are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. In some embodiments, in order to increase the expression of the nucleotide sequence used according to this disclosure, the nucleotide sequence may be modified in the coding region, i.e., within the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, for example, to increase GC content, thereby increasing RNA stability, and / or to perform codon optimization, and thereby enhancing translation in the cell.

[0623] Composition

[0624] In some embodiments, this disclosure also provides a composition comprising the polynucleotides or RNA disclosed herein. In another embodiment, this disclosure also provides a composition comprising the RNA constructs disclosed herein.

[0625] In some embodiments, this disclosure provides a composition comprising RNA or nucleoside-modified RNA encoding a polypeptide, said polypeptide comprising HSV glycoprotein B (gB) or an immunogenic fragment thereof. In some embodiments, the composition comprises RNA or nucleoside-modified RNA encoding a polypeptide, said polypeptide comprising HSV-1 gB or an immunogenic fragment thereof. In another embodiment, the composition comprises RNA or nucleoside-modified RNA encoding a polypeptide, said polypeptide comprising HSV-2 gB or an immunogenic fragment thereof.

[0626] In some embodiments, this disclosure describes compositions, and in some embodiments, the compositions are immunogenic compositions. In some embodiments, the immunogenic compositions used in the compositions and methods described herein are vaccines. In some embodiments, the advantage of RNA technology is that it can include multiple immunogens in the composition (e.g., an immunogenic composition, such as a vaccine), rather than the more limited number of antigens used in protein vaccines. Thus, in some embodiments, in addition to HSV-2 gB or a portion thereof, the polynucleotides as described herein encode one or more additional antigens or immunogens. In another embodiment, in addition to the polynucleotides encoding HSV-2 gB or a portion thereof, the compositions as described herein contain one or more additional polynucleotides encoding one or more additional antigens or immunogens.

[0627] In another embodiment, the additional antigen or immunogen comprises one or more HSV glycoproteins. In some embodiments, the HSV glycoprotein comprises glycoprotein H. In another embodiment, the HSV glycoprotein comprises glycoprotein L. In another embodiment, the HSV glycoprotein comprises glycoprotein I. In another embodiment, the HSV glycoprotein comprises glycoprotein C. In another embodiment, the HSV glycoprotein comprises glycoprotein D. In another embodiment, the HSV glycoprotein comprises glycoprotein E. In another embodiment, the one or more HSV glycoproteins comprise a combination of the glycoproteins described herein. In some embodiments, the additional antigen or immunogen is derived from HSV-1. In another embodiment, the additional antigen or immunogen is derived from HSV-2. The sequences for HSV-2 glycoprotein C, HSV-2 glycoprotein D, and HSV-2 glycoprotein E have been published, see, for example, WO 2019 / 035066, which is incorporated herein by reference in its entirety.

[0628] In some embodiments, in addition to the polynucleotide encoding HSV-2 gB disclosed herein, the compositions disclosed herein further comprise RNA encoding one or more polypeptides encoded by immediate early genes such as ICP4, ICP10, and ICP27. In another embodiment, in addition to the polynucleotide encoding HSV-2 gB disclosed herein, the compositions disclosed herein further comprise RNA encoding one or more cortical structural proteins such as VP11 / 12, VP13 / 14, VP16, and VP22.

[0629] In another embodiment, the compositions disclosed herein (e.g., immunogenic compositions, in some embodiments, said compositions are vaccines) comprise one or more T-cell immunogens. In another embodiment, the compositions disclosed herein comprise immunogens from HSV-1 and HSV-2.

[0630] In some embodiments, this disclosure provides a composition comprising (i) RNA or nucleoside-modified RNA encoding the HSV-2gB or an immunogenic fragment thereof disclosed herein, and (ii) RNA or nucleoside-modified RNA encoding one or more HSV-2 glycoproteins H, L, and I or an immunogenic fragment thereof.

[0631] In another embodiment, this disclosure provides a composition comprising (i) RNA or nucleoside-modified RNA encoding the HSV-2 gB or an immunogenic fragment thereof disclosed herein, and (ii) RNA or nucleoside-modified RNA encoding HSV-2 glycoproteins C, D, and E or immunogenic fragments thereof. Sequences for HSV-2 glycoproteins C, D, and E have been published, see, for example, WO 2019 / 035066, which is incorporated herein by reference in its entirety. In other embodiments, the RNA or nucleoside-modified RNA encoding HSV-2 glycoprotein C or an immunogenic fragment thereof comprises one or more sequences as shown in SEQ ID NO: 259 or 265. In other embodiments, the RNA or nucleoside-modified RNA encoding HSV-2 glycoprotein D or an immunogenic fragment thereof comprises one or more sequences as shown in SEQ ID NO: 261 or 267. In other embodiments, the RNA encoding HSV-2 glycoprotein E or its immunogenic fragments, or the nucleoside-modified RNA, comprises one or more sequences as shown in SEQ ID NO: 263 or 269.

[0632] In other embodiments, the RNA encoding HSV-2 glycoprotein C or its immunogenic fragment, or the nucleoside-modified RNA, encodes a polypeptide as shown in SEQ ID NO: 258 or 264. In other embodiments, the RNA encoding HSV-2 glycoprotein D or its immunogenic fragment, or the nucleoside-modified RNA, encodes a polypeptide as shown in SEQ ID NO: 260 or 266. In other embodiments, the RNA encoding HSV-2 glycoprotein E or its immunogenic fragment, or the nucleoside-modified RNA, encodes a polypeptide as shown in SEQ ID NO: 262 or 268.

[0633] In some embodiments, this disclosure provides a composition comprising (i) RNA or nucleoside-modified RNA encoding HSV-2 gB or an immunogenic fragment thereof disclosed herein, and (ii) RNA encoding one or more polypeptides or immunogenic fragments thereof, said polypeptides being encoded by immediate early genes such as ICP0, ICP4, ICP10, and ICP27. In some embodiments, this disclosure provides a composition comprising (i) RNA or nucleoside-modified RNA encoding HSV-2 gB or an immunogenic fragment thereof disclosed herein, (ii) RNA or nucleoside-modified RNA encoding ICP0 or an immunogenic fragment thereof, and (iii) RNA or nucleoside-modified RNA encoding ICP4 or an immunogenic fragment thereof. In another embodiment, the composition comprises (i) RNA or nucleoside-modified RNA encoding HSV-2 gB or an immunogenic fragment thereof disclosed herein, and (ii) RNA encoding one or more cortical structural proteins such as VP11 / 12, VP13 / 14, VP16, and VP22 or immunogenic fragments thereof.

[0634] In some embodiments, a composition comprising RNA or nucleoside-modified RNA encoding HSV-2 gB or an immunogenic fragment thereof disclosed herein further comprises one or more RNAs or nucleoside-modified RNAs encoding: (a) HSV glycoprotein C (gC) or an immunogenic fragment thereof, (b) HSV glycoprotein D (gD) or an immunogenic fragment thereof, (c) HSV glycoprotein E (gE) or an immunogenic fragment thereof, (d) HSV glycoprotein H (gH) or an immunogenic fragment thereof, (e) HSV glycoprotein L (gL) or an immunogenic fragment thereof, (f) HSV glycoprotein I (gI) or an immunogenic fragment thereof, or (f) any combination thereof.

[0635] In some embodiments, a composition comprising RNA encoding the HSV-2 gB or an immunogenic fragment thereof disclosed herein, or nucleoside-modified RNA, further comprises nanoparticles, lipids, polymers, cholesterol, or cell-penetrating peptides. In some embodiments, the nanoparticles may be lipid nanoparticles. In some embodiments, the nanoparticles, lipids, polymers, cholesterol, or cell-penetrating peptides encapsulate the polynucleotide or RNA constructs disclosed herein.

[0636] In some embodiments, one or more polynucleotides are encapsulated in nanoparticles. In some embodiments, one or more polynucleotides are encapsulated in lipids. In some embodiments, one or more polynucleotides are encapsulated in polymers. In some embodiments, one or more polynucleotides are encapsulated in cholesterol. In some embodiments, one or more polynucleotides are encapsulated in cell-penetrating peptides.

[0637] In some embodiments, one or more polynucleotides are attached to nanoparticles. In some embodiments, one or more polynucleotides are attached to lipids. In some embodiments, one or more polynucleotides are attached to polymers. In some embodiments, one or more polynucleotides are attached to cholesterol. In some embodiments, one or more polynucleotides are attached to cell-penetrating peptides.

[0638] In some embodiments, the method of this disclosure further includes mixing the RNA with the transfection reagent prior to the contact step. In another embodiment, the method of this disclosure further includes applying the RNA together with the transfection reagent. In yet another embodiment, the transfection reagent is a cationic lipid reagent.

[0639] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin® or Lipofectamine®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0640] In another embodiment, the transfection reagent forms liposomes. In yet another embodiment, liposomes enhance intracellular stability, increase uptake efficiency, and improve biological activity.

[0641] In another embodiment, a liposome is a hollow, spherical vesicle composed of lipids arranged in a manner similar to that of lipids constituting a cell membrane. In another embodiment, the liposome has an internal aqueous space for capturing water-soluble compounds and a diameter ranging from 0.05 micrometers to several micrometers. In another embodiment, liposomes can deliver RNA to cells in a biologically active form (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0642] Each type of transfection reagent represents a separate embodiment of this disclosure.

[0643] Liposomes are known to enhance intracellular stability, increase uptake efficiency, and improve biological activity. In some embodiments, liposomes are hollow, spherical vesicles composed of lipids arranged in an arrangement similar to that of lipids constituting a cell membrane. In another embodiment, the liposomes have internal aqueous spaces for capturing water-soluble compounds and have a diameter ranging from 0.05 micrometers to several micrometers. In yet another embodiment, liposomes can deliver RNA to cells in a biologically active form.

[0644] Nanoparticles and lipid nanoparticles

[0645] In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more nanoparticles. In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more lipids. In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more polymers. In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more cholesterol. In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more cell-penetrating peptides. In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more of the above.

[0646] In some embodiments, in addition to polynucleotides, the compositions or combinations described herein further comprise any combination of one or more nanoparticles, lipids, polymers, cholesterol, and cell-penetrating peptides.

[0647] In some embodiments, the polynucleotide is associated with one or more nanoparticles. In some embodiments, the polynucleotide is associated with one or more lipids. In some embodiments, the polynucleotide is associated with one or more polymers. In some embodiments, the polynucleotide is associated with one or more cholesterol molecules. In some embodiments, the polynucleotide is associated with one or more cell-penetrating peptides. In some embodiments, in addition to the polynucleotide, the compositions described herein further comprise one or more of the above.

[0648] In some embodiments, the polynucleotide is attached to one or more nanoparticles. In some embodiments, the polynucleotide is attached to one or more lipids. In some embodiments, the polynucleotide is attached to one or more polymers. In some embodiments, the polynucleotide is attached to one or more cholesterol molecules. In some embodiments, the polynucleotide is attached to one or more cell-penetrating peptides. In some embodiments, in addition to polynucleotides, the compositions described herein further comprise one or more of the above.

[0649] In some embodiments, the polynucleotide is encapsulated in one or more nanoparticles. In some embodiments, the polynucleotide is encapsulated in one or more lipids. In some embodiments, the polynucleotide is encapsulated in one or more polymers. In some embodiments, the polynucleotide is encapsulated in one or more cholesterol molecules. In some embodiments, the polynucleotide is encapsulated in one or more cell-penetrating peptides.

[0650] In some embodiments, the nanoparticles used in the compositions and methods of this disclosure comprise one or more lipid nanoparticles, such as Cullis, P. and Hope, M. (date unknown), enabling Lipid Nanoparticle Systems for Enabling Gene Therapies, as described in Molecular Therapy, 25(7), which is incorporated herein by reference in its entirety.

[0651] In some embodiments, the delivery of RNA (e.g., nucleoside-modified RNA) includes any suitable delivery method, including exemplary RNA transfection methods described elsewhere herein. In some embodiments, delivering RNA or nucleoside-modified RNA to a subject includes mixing the RNA with a transfection reagent prior to the contact step. In another embodiment, the method of this disclosure further includes administering the RNA together with the transfection reagent. In yet another embodiment, the transfection reagent is a cationic lipid reagent.

[0652] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0653] In another embodiment, the transfection reagent forms liposomes.

[0654] In another embodiment, liposomes enhance intracellular stability, increase uptake efficiency, and improve biological activity. In another embodiment, a liposome is a hollow, spherical vesicle composed of lipids arranged in an arrangement similar to that of lipids constituting a cell membrane. In another embodiment, the liposome has an internal aqueous space for capturing water-soluble compounds and a diameter ranging from 0.05 micrometers to several micrometers. In another embodiment, liposomes can deliver RNA to cells in a biologically active form.

[0655] In some embodiments, the composition comprises lipid nanoparticles (LNPs) as described herein and one or more nucleic acid molecules. For example, in some embodiments, the composition comprises an LNP and one or more RNA or nucleoside-modified RNA molecules encoding one or more antigens, adjuvants, or combinations thereof.

[0656] The term “lipid nanoparticle” refers to at least one particle with a size in the nanometer range (e.g., 1-1,000 nm), the particle comprising one or more lipids, such as lipids of formula (I), (II) or (III) as described in WO 2016176330A1, which is incorporated herein by reference in its entirety.

[0657] In some embodiments, lipid nanoparticles are included in formulations containing RNA or nucleoside-modified RNA as described herein. In some embodiments, such lipid nanoparticles comprise cationic lipids and one or more excipients selected from: neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., PEGylated lipids, such as PEGylated lipids of structure (IV), such as compound IVa). In some embodiments, RNA or nucleoside-modified RNA is encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects, such as adverse immune responses, induced by mechanisms of the host organism or cells.

[0658] In various embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In some embodiments, when present in lipid nanoparticles, the RNA or nucleoside-modified RNA is resistant to degradation by nucleases in aqueous solution.

[0659] LNPs can comprise any lipid capable of forming particles to which one or more nucleic acid molecules are attached or encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids,” such as steroids.

[0660] In some embodiments, the LNP comprises one or more cationic lipids and one or more stable lipids. Stable lipids include neutral lipids and PEGylated lipids.

[0661] In some embodiments, the LNP comprises cationic lipids. As used herein, the term "cationic lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH decreases below the pK of the lipid's ionizable group, but gradually becomes more neutral at higher pH values. At pH values ​​below the pK, the lipid is then able to associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises zwitterionic lipids that exhibit a positive charge as the pH decreases.

[0662] In some embodiments, cationic lipids comprise any of a variety of lipid species carrying a net positive charge at a selectable pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearate-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP); and 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi). The following are examples of amino acids disclosed in this disclosure: N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarbamoyl)ethyl-N,N-dimethyltrifluoroacetate ammonium (DOSPA), octadecylamidoglycosylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), and N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Furthermore, commercial formulations of various cationic lipids disclosed herein are available. These include, for example, LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (commercially available cationic liposomes containing N-(1-(2,3-dioleoyloxy)propyl)-N-(2-(sperminecarbamoyl)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids containing octadecylamidoglycosylcarboxyspermine (DOGS) in ethanol, from Promega Corp., Madison, Wisconsin). The following lipids are cationic and positively charged at pH below physiological levels:

[0663] DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenicoxy-N,N-dimethylaminopropane (DLenDMA).

[0664] In some embodiments, the cationic lipid is an amino lipid. Suitable amino lipids that can be used in this disclosure include those described in WO 2012 / 016184, which is incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), and 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl). 2-Linoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA) and 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0665] In some embodiments, cationic lipids are present in the LNP in an amount from about 30 mol% to about 95 mol%. In some embodiments, cationic lipids are present in the LNP in an amount from about 30 mol% to about 70 mol%. In some embodiments, cationic lipids are present in the LNP in an amount from about 40 mol% to about 60 mol%. In some embodiments, cationic lipids are present in the LNP in an amount from about 50 mol%. In some embodiments, the LNP contains only cationic lipids. In some embodiments, the LNP contains one or more additional lipids that stabilize particle formation during particle formation.

[0666] Suitable stable lipids include neutral lipids and anionic lipids.

[0667] The term "neutral lipid" refers to any of the many lipid species that exist as uncharged or neutral zwitterions at physiological pH.

[0668] Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0669] Exemplary neutral lipids include, for example, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimide). The neutral lipids are 1,2-distearyl-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC).

[0670] In some embodiments, the LNP comprises neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipids (e.g., lipids of formula (I)) to neutral lipids ranges from about 2:1 to about 8:1.

[0671] In various embodiments, the LNP further comprises a steroid or a steroid analogue.

[0672] In some embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of cationic lipid (e.g., lipid of formula (I)) to cholesterol ranges from about 2:1 to 1:1.

[0673] The term "anionic lipid" refers to any lipid that carries a negative charge at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups conjugated to neutral lipids.

[0674] In some embodiments, the LNP comprises glycolipids (e.g., monosialotetrahexosylganglioside GMi). In some embodiments, the LNP comprises sterols, such as cholesterol.

[0675] In some embodiments, the LNP comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), etc.

[0676] In some embodiments, the LNP comprises additional stabilizing lipids, which are polyethylene glycol-lipids (PEGylated lipids). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

[0677] Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In some embodiments, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol-)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP comprises PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEGylated succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecyloxy)propyl)-1-O-(comethoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEGylated dialkoxypropyl carbamate, such as Q-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(comethoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0678] In some embodiments, additional lipids are present in the LNP in an amount from about 1 mole percent to about 10 mole percent. In some embodiments, additional lipids are present in the LNP in an amount from about 1 mole percent to about 5 mole percent. In some embodiments, additional lipids are present in the LNP in an amount from about 1 mole percent to about 1.5 mole percent.

[0679] In some embodiments, the LNP includes one or more targeting portions capable of targeting the LNP to cells or cell populations. For example, in some embodiments, the targeting portion is a ligand that directs the LNP to a receptor present on the cell surface.

[0680] In some embodiments, the LNP comprises one or more internalization domains. For example, in some embodiments, the LNP comprises one or more domains that bind to cells to induce LNP internalization. For example, in some embodiments, one or more internalization domains bind to receptors present on the cell surface to induce receptor-mediated uptake of the LNP. In some embodiments, the LNP is capable of binding to biomolecules in vivo, wherein the biomolecule bound to the LNP can then be recognized by cell surface receptors to induce internalization. For example, in some embodiments, the LNP binds to systemic ApoE, which induces uptake of the LNP and associated cargo.

[0681] Other exemplary LNPs and their preparation are described in the art, for example in WO 2016176330A1, U.S. Patent Application Publication No. US20120276209; Semple et al., 2010, *Nature Biotechnol*, 28(2): 172-176; Akinc et al., 2010, *Molecular Therapy*, 18(7): 1357-1364; Basha et al., 2011, *Molecular Therapy*, 19(12): 2186-2200; Leung et al., 2012, *Journal of Physical Chemistry C: Nanomaterials and Interfaces*, 116(34): 18440-18450; Lee et al., 2012, *International Journal of Cancer*, 131(5): E781-90; Belliveau et al., 2012, Molecular Therapy Nucleic Acids, 1: e37; Jayaraman et al., 2012, Angewandte Chemie International Edition, 51(34): 8529-8533; Mui et al., 2013, Molecular Therapy Nucleic Acids 2, el39; Maier et al., 2013, Molecular Therapy, 21(8): 1570-1578; and Tarn et al., 2013, Nanomedicine, 9(5): 665-74, each of which is incorporated herein by reference in its entirety.

[0682] In another embodiment, the method of this disclosure comprises administering RNA encoding one or more HSV glycoproteins or immunogenic fragments thereof, along with a pharmaceutically acceptable carrier or diluent. In other embodiments, the pharmaceutically acceptable carrier for the liquid formulation may be an aqueous or non-aqueous solution, a suspension, an emulsion, or an oil. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Examples of oils are petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and cod liver oil.

[0683] As used herein, "pharmaceutically acceptable carrier or diluent" is well known to those skilled in the art.

[0684] In another embodiment, the pharmaceutical composition provided herein is a sustained-release composition, i.e., a composition that releases the compound over a period of time after administration. Controlled-release or sustained-release compositions comprise formulations in a lipophilic library (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e., a composition that releases the entire compound immediately after administration.

[0685] Each of the additives, excipients, formulations, and methods of application represents a separate embodiment of this disclosure.

[0686] In another embodiment, this disclosure provides a kit containing reagents used in performing the methods of this disclosure. In yet another embodiment, this disclosure provides a kit comprising the compositions, tools, or instruments of this disclosure.

[0687] peptides and host cells

[0688] This disclosure also provides a polypeptide encoded by the polynucleotide or RNA construct disclosed herein. In some embodiments, the polypeptide comprises an extracellular domain of HSV-1 gB or an immunogenic fragment thereof, and in some embodiments, the extracellular domain or immunogenic fragment thereof is as shown in SEQ ID NO: 10.

[0689] In another embodiment, the polypeptide comprises an extracellular domain of HSV-2 gB or an immunogenic fragment thereof. In some embodiments, the extracellular domain of HSV-2 gB comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extracellular domain of HSV-2 gB consists of or is substantially composed of the amino acid sequence of SEQ ID NO: 1. In some embodiments, this disclosure covers a sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1.

[0690] This disclosure also provides a host cell comprising a polypeptide encoded by the polynucleotide or RNA construct disclosed herein. In some embodiments, the host cell is a human cell.

[0691] Treatment methods and the use of compounds and compositions

[0692] This disclosure also provides a method for treating a subject with herpes simplex virus (HSV) infection, the method comprising administering the disclosed polynucleotide to the subject. In another embodiment, use of the disclosed polynucleotide is provided for the preparation of a medicament for treating HSV infection. This disclosure also covers a disclosed polynucleotide for treating HSV infection.

[0693] This disclosure also provides a method for treating a subject with herpes simplex virus (HSV) infection, the method comprising the step of administering a composition to the subject, the composition comprising the polynucleotide or RNA construct disclosed herein. In another embodiment, use of the disclosed composition is provided for preparing a medicament for treating HSV infection. This disclosure also covers a disclosed composition for treating HSV infection.

[0694] In another embodiment, this disclosure provides a method for treating HSV infection in a subject, the method comprising administering to the subject a polynucleotide or nucleoside-modified polynucleotide encoding a polypeptide or composition, wherein the polynucleotide or nucleoside-modified polynucleotide encodes a polypeptide or nucleoside-modified polynucleotide, wherein the polypeptide encodes an HSV gB antigen or an immunogenic fragment thereof. In another embodiment, this disclosure provides a method for treating HSV infection in a subject, the method comprising administering to the subject a polynucleotide or nucleoside-modified polynucleotide encoding a polypeptide or composition, wherein the polynucleotide or nucleoside-modified polynucleotide encodes a polypeptide or nucleoside-modified polynucleotide, wherein the polypeptide encodes an HSV-2 gB antigen or an immunogenic fragment thereof. In another embodiment, this disclosure provides a method for treating HSV infection in a subject, the method comprising administering to the subject a polynucleotide or nucleoside-modified polynucleotide encoding a polypeptide or ...

Claims

1. A nucleoside-modified polynucleotide encoding a polypeptide, wherein the polypeptide comprises an extracellular domain of the herpes simplex virus-2 (HSV-2) glycoprotein B (gB) antigen, the extracellular domain having an amino acid sequence as shown in SEQ ID NO:

1.

2. The polynucleotide according to claim 1, wherein the nucleoside modification comprises one or more pseudouridine residues.

3. The polynucleotide according to claim 2, wherein one or more pseudouridine residues comprise m1Ψ (1-methylpseudouridine).

4. The polynucleotide according to claim 2, wherein the one or more pseudouridine residues comprise m 1 acp 3 Ψ (1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine), Ψm (2'-O-methylpseudouridine), m 5 D (5-methyldihydrouridine), m 3 Ψ (3-methylpseuuridine) or any combination thereof.

5. The polynucleotide according to any one of claims 1 to 4, further comprising a secretion signal sequence.

6. The polynucleotide according to any one of claims 1 to 5, comprising the ribonucleic acid sequence shown in SEQ ID NO:

5.

7. The polynucleotide according to claim 5, wherein the secretion signal sequence encodes an IL2 secretion signaling polypeptide.

8. The polynucleotide of claim 7, wherein the IL2 secretion signaling polypeptide comprises the amino acid sequence shown in SEQ ID NO:

2.

9. The polynucleotide of claim 7, wherein the IL2 secretion signaling polypeptide is encoded by a ribonucleic acid sequence as shown in SEQ ID NO:

3.

10. The polynucleotide of claim 5, wherein the secretion signal sequence encodes the HSV gB secretion signal polypeptide.

11. The polynucleotide of claim 10, wherein the gB secretion signaling polypeptide comprises the amino acid sequence shown in SEQ ID NO:

4.

12. The polynucleotide according to any one of claims 1 to 11, wherein the polynucleotide further comprises a 5' untranslated region.

13. The polynucleotide according to any one of claims 1 to 12, wherein the polynucleotide further comprises a cap-independent translation enhancer.

14. The polynucleotide according to any one of claims 1 to 13, wherein the polynucleotide further comprises a 3' untranslated region.

15. The polynucleotide according to any one of claims 1 to 14, wherein the polynucleotide further comprises a polyA tail.

16. The polynucleotide according to any one of claims 1 to 15, wherein the polynucleotide further comprises an m7GpppG cap, a 3'-O-methyl-m7GpppG cap, or an anti-reverse cap analogue.

17. The polynucleotide according to any one of claims 1 to 9 or 12 to 16, comprising a ribonucleic acid sequence as shown in SEQ ID NO:

6.

18. A composition or combination comprising a polynucleotide according to any one of claims 1 to 17.

19. The composition or combination of claims 18, wherein the composition comprises nanoparticles, lipids, polymers, cholesterol, or cell-penetrating peptides.

20. The composition or combination of claim 19, wherein the nanoparticles, the lipids, the polymers, the cholesterol, or the cell-penetrating peptides encapsulate polynucleotides.

21. The composition or combination according to claim 20, wherein the nanoparticles are lipid nanoparticles.

22. The composition or combination according to any one of claims 18 to 21, further comprising one or more RNAs encoding the following: a) HSV glycoprotein C (gC) or its immunogenic fragment, b) HSV glycoprotein D (gD) or its immunogenic fragment, c) HSV glycoprotein E (gE) or its immunogenic fragment, d) HSV glycoprotein H (gH) or its immunogenic fragment, e) HSV glycoprotein L (gL) or its immunogenic fragment, f) HSV glycoprotein I (gI) or its immunogenic fragment, or g) Any combination thereof.

23. A polypeptide encoded by a polynucleotide according to any one of claims 1 to 17.

24. A host cell comprising a polynucleotide according to any one of claims 1 to 17.

25. A host cell comprising the polypeptide according to claim 23.

26. A method for treating a subject with herpes simplex virus (HSV) infection, the method comprising administering to the subject a polynucleotide according to any one of claims 1 to 17 or a composition or combination according to any one of claims 18 to 22.

27. A method for inhibiting or reducing the incidence of herpes simplex virus (HSV) infection in a subject, the method comprising administering to the subject a polynucleotide according to any one of claims 1 to 17 or a composition or combination according to any one of claims 18 to 22.

28. The method of claim 26 or 27, wherein the HSV infection comprises HSV-1 infection.

29. The method of claim 26 or 27, wherein the HSV infection comprises HSV-2 infection.

30. The method of claim 26 or 27, wherein the HSV infection is a primary or secondary infection.

31. The method of claim 26 or 27, wherein the HSV infection comprises sudden onset, recurrence, HSV cold sores following primary HSV infection, reactivation of latent HSV infection, HSV encephalitis, HSV neonatal infection, genital HSV infection, oral HSV infection, or a combination thereof.

32. The method of claim 26 or 27, wherein the HSV infection is a latent HSV infection.

33. The method of claim 32, wherein the latent HSV infection comprises genital HSV infection or oral HSV infection.

34. The method of claim 26 or 27, wherein the application comprises intramuscular, subcutaneous, intradermal, intranasal, intravaginal, intrarectal, or local application.

35. A method for inducing an anti-HSV immune response in a subject, the method comprising administering to the subject a polynucleotide according to any one of claims 1 to 17 or a composition or combination according to any one of claims 18 to 22.

36. The method of claim 35, wherein the immune response comprises a CD4 immune response, a CD8 immune response, a T follicular helper cell immune response, a germinal center B cell immune response, an IgG antibody response against gB2, or a combination thereof.

37. The method of claim 35, wherein the application comprises intramuscular, subcutaneous, intradermal, intranasal, intravaginal, intrarectal, or local application.

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