Modified mRNA vaccines encoding herpes simplex virus glycoproteins and uses thereof

Abstract

To provide modified mRNA vaccines encoding herpes simplex virus glycoproteins and uses thereof.SOLUTION: The invention provides compositions comprising nucleoside-modified mRNAs encoding herpes simplex virus (HSV) glycoproteins including those involved in virus entry and immune evasion for preventing and treating genital herpes and uses thereof. In one embodiment, the invention provides compositions comprising one or more nucleoside-modified mRNAs, where each of the nucleoside-modified mRNA encodes a glycoprotein of HSV or immunogenic fragment thereof, and the nucleoside-modified mRNA comprises one or more pseudouridine residues.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention provides a composition for the prevention and treatment of genital herpes, comprising a nucleoside-modified mRNA encoding a herpes simplex virus (HSV) glycoprotein, including those involved in virus entry and immune evasion, and a method of using the same.

Background Art

[0002] Genital herpes vaccines are urgently desired to prevent pain and suffering, reduce the incidence of neonatal herpes, and lower the risk of transmission associated with HIV acquisition and genital infections. Approximately 500 million people worldwide are infected with herpes simplex virus type 2 (HSV-2), the virus that causes genital herpes. In some individuals, the infection results in painful recurrent genital ulcers, while in others, the infection remains asymptomatic. In both settings, infected individuals can transmit the virus to their intimate partners. Genital herpes increases the risk that an infected person will acquire HIV if exposed during sexual intercourse. A vaccine for genital herpes is urgently needed and none are currently available.

[0003] Chiron Corp. investigated a prophylactic vaccine containing glycoprotein B (gB2) and D (gD2), two HSV-2 glycoproteins involved in virus entry, given with MF59 as an adjuvant. This vaccine delayed the onset of infection for the first five months after immunization, but seronegative partners were not protected from HSV-2 infection. GlaxoSmithKline (GSK) ​, a prophylactic vaccine using the gD2 antigen containing monophosphoryl lipid A (MPL) and alum as an adjuvant was evaluated. Overall, significant protection was observed in a subgroup of women who were doubly seronegative for HSV-1 and HSV-2, but protection against genital lesions was not detected. A follow-up study was conducted on doubly seronegative women who did not show overall protection against genital herpes; however, the vaccine was effective against HSV-1. This result was notable because HSV-1 accounted for 60% of genital herpes infections in the control group. These studies indicate that they were not sufficient to target a vaccine that blocks HSV-2 entry.

[0004] The gC of HSV-1 and HSV-2 are immune evasion molecules that function as regulators of the complement cascade. During complement activation, the most abundant complement protein C3 is cleaved into C3b, activating a membrane-damaging complex that leads to virus neutralization and lysis of infected cells. C3b stimulates B-cell and T-cell responses and functions as a link between innate and acquired immunity. The gC of HSV-1 and HSV-2 binds C3b and inhibits C3b-mediated activities. Immunization with gC1 and gC2 produces antibodies that bind to the glycoprotein and block its immune evasion function.

[0005] The glycoprotein E (gE) of HSV-1 and HSV-2 functions as an immune evasion molecule by binding to the Fc domain of IgG molecules that bind to its target by their F(ab’)2 domain. A vaccine containing the gE2 subunit antigen produces antibodies that bind to gE2 and block its immune evasion function. The gC2 and gE2 of HSV-2 perform activities similar to those of mammalian complement and IgG Fc regulatory proteins, but do not share sequence homology with mammalian receptors, which virtually suggests that immunization does not carry the risk of inducing autoimmunity.

[0006] In previous studies from our laboratory, vaccines containing gC, gD, and gE have been tested and found to provide protection against HSV infection. However, it is not known whether mRNA vaccines encoding HSV gC, gD, and gE will be effective in providing protection against HSV infection.

[0007] There are several advantages to using nucleic acids as vaccines. Nucleic acid vaccines can induce both humoral and cellular immune responses; have a low effective dose; are easy to handle; are useful for rapid testing; are cost-effective and reproducible in terms of large-scale production and isolation; can be produced frequently and are easy to isolate; are more temperature stable than conventional vaccines; have a long storage period; are easy to store and transport; and do not seem to require a cold chain (Shedlock & Weiner, J Leukocyte Biol. Vol 68, 2000).

[0008] In principle, any foreign DNA or RNA can express proteins in the mammalian body. It is unclear whether similar immune activities can be generated using both proteins by DNA and mRNA expression. DNA is a dogma that is superior to vaccine production and gene therapy due to its stability and ease of use.

[0009] DNA has been successfully used in vaccines. DNA is quite stable and non-reactive and can be stored for a long time. However, DNA can self-replicate and be easily damaged by ultraviolet irradiation. DNA-based vaccines can also raise concerns about safety due to possible insertion of DNA into the genome, possible interruption of genes, and formation of anti-DNA antibodies.

[0010] RNA vaccines exhibit important safety features. RNA is more reactive and less stable than DNA, but is resistant to ultraviolet irradiation. mRNA is not incorporated into the host chromosome. Delivery of mRNA results in faster expression of the antigen of interest, and fewer copies are required for expression. mRNA expression is transient, which enhances its safety. mRNA is more effective than DNA for protein production in post-mitotic and non-dividing cells because, while DNA requires translocation through the nuclear membrane and the plasma membrane, mRNA only requires translocation through the plasma membrane. mRNA serves not only as a template for translation but also acts as a ligand for Toll-like receptors and is nuclease-sensitive; therefore, there are fewer concerns regarding horizontal transmission. Furthermore, RNA vaccines elegantly incorporate adjuvant activity and antigen expression, thereby mimicking aspects relevant to viral infection. This increases the efficacy of RNA vaccines compared to inactivated vaccines that require the use of adjuvants, and simplifies handling and production. RNA can engage a range of specialized immune pattern recognition receptors, including Toll-like receptors 3, 7, and 8, RIG-I, MDA5, PKR, and other receptors that can act synergistically to enhance the induction of antigen-specific adaptive B cell and T cell responses. Importantly, antigen synthesis in transfected host cells directly introduces the antigen into the processing and presentation pathway of cellular antigens by mRNA vaccines, establishing access to MHC molecules and inducing a T cell response regardless of the host MHC haplotype. This enables the induction of a polyclonal T cell response that can act synergistically with other immune responses, including B cells. Additionally, endogenous antigen production ensures faithful post-translational modifications (e.g., protein processing, glycosylation, etc.) that can positively impact immunogenicity.

Prior Art Documents

Non-Patent Documents

[0011]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0012] In one embodiment, the present invention provides a composition comprising one or more nucleoside-modified mRNAs, wherein each of the nucleoside-modified mRNAs encodes a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and the nucleoside-modified mRNA contains one or more pseudouridine residues.

[0013] In another embodiment, the present invention provides a composition comprising one or more nucleoside-modified mRNAs, wherein each of the modified mRNAs encodes a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and the nucleoside-modified mRNA contains 1-methylpseudouridine. The pseudouridine residue is m 1 acp 3 Ψ(1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, m 1 Ψ(1-methylpseudouridine), Ψm(2'-O-methylpseudouridine, m 5 D(5-methyldihydrouridine), m 3 Ψ(3-methylpseudouridine), or any combination thereof.

[0014] In another embodiment, the present invention provides a composition comprising modified mRNAs containing one or more pseudouridine residues, wherein each of the modified mRNAs encodes a) herpes simplex virus glycoprotein D (gD) or an immunogenic fragment thereof, b) herpes simplex virus glycoprotein C (gC) or an immunogenic fragment thereof, c) herpes simplex virus glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof.

[0015] In another embodiment, the present invention provides a method for treating herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof, and the modified mRNAs comprising pseudouridine residues.

[0016] In another embodiment, the present invention provides a method for inducing an immune response in a subject, comprising administering to the subject a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof, and the modified mRNAs comprising pseudouridine residues.

[0017] In a further embodiment, the present invention provides a method for suppressing, inhibiting, or reducing the occurrence of herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof, and the modified mRNAs comprising pseudouridine residues.

[0018] In yet another embodiment, the present invention provides a method for treating herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition comprising one or more modified mRNAs encoding a) herpes simplex virus glycoprotein D (gD) or an immunogenic fragment thereof, b) herpes simplex virus glycoprotein C (gC) or an immunogenic fragment thereof, c) herpes simplex virus glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof.

[0019] In yet a further embodiment, the present invention provides a method for suppressing, inhibiting, or reducing the occurrence of herpes simplex virus (HSV) infection in a subject, the method comprising administering to the subject a composition comprising one to three modified mRNAs, each of the modified mRNAs encoding a) herpes simplex virus glycoprotein D (gD) or an immunogenic fragment thereof, b) herpes simplex virus glycoprotein C (gC) or an immunogenic fragment thereof, and c) herpes simplex virus glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof.

[0020] Other features and advantages of the present invention will become apparent from the examples and drawings in the following detailed description of the invention. However, it should be understood by those skilled in the art that various changes and modifications within the spirit and scope of the invention will become apparent from this detailed description of the invention. Therefore, the detailed description of the invention and the specific examples are provided for illustrative purposes only while showing the preferred embodiments of the invention.

Brief Description of the Drawings

[0021] The following drawings form a part of this specification and are included to further demonstrate specific aspects of the present disclosure. The invention can be better understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments shown herein. The patent or application file contains at least one drawing created in color. Copies of this patent or application publication, including the color drawing(s), will be provided by the Patent Office upon payment of the necessary fees for requesting the same.

[0022]

Figure 1

[0023] Figure 1A is a Western blot showing the expression of gC2 by the modified mRNA.

[0024] Figure 1B is a Western blot showing the expression of gD2 by the modified mRNA.

[0025] Figure 1C is a Western blot showing the expression of gE2 by the modified mRNA.

[0026]

Figure 2

[0027] Figure 2B shows the gD2 Ab response determined by antigen - specific ELISA in mice immunized with gD2 mRNA; or gC2 mRNA, gD2 mRNA, and gE2 mRNA (trivalent - I) each given at different intradermal sites; or gC2 mRNA, gD2 mRNA, and gE2 mRNA (trivalent - C) given in combination. I indicates the first immunization; II indicates the second immunization.

[0028] Figure 2C shows the gE2 Ab response determined by antigen - specific ELISA in mice immunized with gD2 mRNA; or gC2 mRNA, gD2 mRNA, and gE2 mRNA (trivalent - I) each given at different intradermal sites; or gC2 mRNA, gD2 mRNA, and gE2 mRNA (trivalent - C) given in combination. I indicates the first immunization; II indicates the second immunization.

[0029]

Figure 3

[0030] Figure 3B shows the antigen-specific IgG2a response in mRNA-vaccinated mice. For the IgG2a response, antibodies were evaluated after the first and second immunizations. I indicates the first immunization; II indicates the second immunization.

[0031]

Figure 4

[0032]

Figure 5

[0033]

Figure 6

[0034]

Figure 7

[0035] Figure 7B shows the weight loss (-) or gain (+) and neurological signs of BALB / c mice immunized twice with mRNA at 28-day intervals and challenged intravaginally with HSV-2. Trivalent-I represents animals immunized with gC2 / LNP, gD2 / LNP, and gE2 / LNP, each administered at different intradermal sites. Trivalent-C represents animals immunized with gC2, gD2, and gE2 formulated in a single LNP for immunization.

[0036]

Figure 8

[0037]

Figure 9

[0038]

Figure 10

[0039]

Figure 11

[0040] In Figure 11B, the trivalent mRNA-LNP vaccine induces a strong germinal center B cell response in mice. BALB / c female mice were left unimmunized as naive control animals or were immunized intradermally twice at 28-day intervals with multivalent C mRNA-LNP or trivalent modified mRNA-LNP. The multivalent C mRNA control received 10 μg of multivalent C mRNA-LNP divided into 4 aliquots and administered at 4 separate sites. The trivalent modified mRNA group received 10 μg of gC2 mRNA-LNP, 10 μg of gD2 mRNA-LNP, and 10 μg of gE2 mRNA-LNP, each divided into 2 aliquots and given at 2 separate sites. Two weeks after the second immunization, spleens were collected from 5 animals per group and flow cytometry was performed to detect the germinal center B cell response (* p < 0.05).

[0041]

Figure 12

[0042]

Figure 13

[0043]

Figure 14-1

Figure 14-2

[0044]

Figure 15

Mode for Carrying Out the Invention

[0045] Composition In one embodiment, the present invention provides a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof.

[0046] In one embodiment, the present invention provides a composition comprising one or more nucleoside-modified mRNAs, each of the modified mRNAs encoding a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and the modified mRNA containing one or more pseudouridines or pseudouridine family residues.

[0047] In one embodiment, the HSV glycoprotein comprises glycoprotein D (gD), glycoprotein C (gC), glycoprotein E (gE), glycoprotein B (gB), glycoprotein H (gH), glycoprotein L (gL), glycoprotein I (gI), or a combination thereof.

[0048] That is, in one embodiment, the present invention provides a composition comprising one or more modified mRNAs encoding gD, gC, gE, gB, gH, gL, gI of HSV, or immunogenic fragments thereof. In one embodiment, the modified mRNA comprises pseudouridine-modified mRNA.

[0049] In one embodiment, the present invention provides a composition comprising a modified mRNA encoding gD of HSV or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gC of HSV or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gE of HSV or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gB of HSV or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gH of HSV or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gL of HSV or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gI of HSV or a fragment thereof.

[0050] In one embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV or a fragment thereof, and (b) a modified mRNA encoding gC of HSV or a fragment thereof.

[0051] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV or a fragment thereof, and (b) a modified mRNA encoding gE of HSV or a fragment thereof.

[0052] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gC of HSV or a fragment thereof, and (b) a modified mRNA encoding gE of HSV or a fragment thereof.

[0053] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV or a fragment thereof, (b) a modified mRNA encoding gC of HSV or a fragment thereof, and (c) a modified mRNA encoding gE of HSV or a fragment thereof.

[0054] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV or a fragment thereof, (b) a modified mRNA encoding gC of HSV or a fragment thereof, (c) a modified mRNA encoding gE of HSV or a fragment thereof, and (d) a modified mRNA encoding gB of HSV or a fragment thereof.

[0055] In one embodiment, the HSV glycoprotein is a HSV-2 glycoprotein. In another embodiment, the HSV glycoprotein is a HSV-1 glycoprotein. In one embodiment, the HSV glycoprotein comprises both HSV-2 glycoprotein and HSV-1 glycoprotein. In another embodiment, the HSV glycoprotein comprises a mixture of HSV-2 glycoprotein and HSV-1 glycoprotein.

[0056] In one embodiment, the present invention provides a composition comprising a modified mRNA encoding gD of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gC of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gE of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gE of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gB of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gH of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gL of HSV-2 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gI of HSV-2 or a fragment thereof.

[0057] In one embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV-2 or a fragment thereof, and (b) a modified mRNA encoding gC of HSV-2 or a fragment thereof.

[0058] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV-2 or a fragment thereof, and (b) a modified mRNA encoding gE of HSV-2 or a fragment thereof.

[0059] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gC of HSV-2 or a fragment thereof, and (b) a modified mRNA encoding gE of HSV-2 or a fragment thereof.

[0060] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV-2 or a fragment thereof, (b) a modified mRNA encoding gC of HSV-2 or a fragment thereof, and (c) a modified mRNA encoding gE of HSV-2 or a fragment thereof.

[0061] In one embodiment, the present invention provides a composition comprising a modified mRNA encoding gD of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gC of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gE of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gE of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gB of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gH of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gL of HSV-1 or a fragment thereof. In another embodiment, the present invention provides a composition comprising a modified mRNA encoding gI of HSV-1 or a fragment thereof.

[0062] In one embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV-1 or a fragment thereof, and (b) a modified mRNA encoding gC of HSV-1 or a fragment thereof.

[0063] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV-1 or a fragment thereof, and (b) a modified mRNA encoding gE of HSV-1 or a fragment thereof.

[0064] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gC of HSV-1 or a fragment thereof, and (b) a modified mRNA encoding gE of HSV-1 or a fragment thereof.

[0065] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding gD of HSV-1 or a fragment thereof, (b) a modified mRNA encoding gC of HSV-1 or a fragment thereof, and (c) a modified mRNA encoding gE of HSV-1 or a fragment thereof.

[0066] In one embodiment, any of the compositions described herein consists essentially of one or more modified mRNAs, each of which encodes an HSV glycoprotein or an immunogenic fragment thereof. In another embodiment, any of the compositions described herein consists of one or more modified mRNAs, each of which encodes an HSV glycoprotein or an immunogenic fragment thereof.

[0067] In another embodiment, the invention provides a composition comprising a modified mRNA encoding an HSV gD protein, a modified mRNA encoding an HSV gC protein, a modified mRNA encoding an HSV gE protein, and a modified mRNA encoding one or more additional HSV glycoproteins. In one embodiment, the additional HSV glycoprotein comprises gB or an immunogenic fragment thereof, gH or an immunogenic fragment thereof, gL or an immunogenic fragment thereof, gI or an immunogenic fragment thereof, or any combination thereof. In one embodiment, the additional HSV glycoprotein comprises glycoprotein M (gM), glycoprotein N (gN), glycoprotein K (gK), glycoprotein G (gG), glycoprotein J (gJ), or an immunogenic fragment thereof.

[0068] In one embodiment, the compositions used in the present invention and the methods of the present invention include both an HSV-2 glycoprotein or glycoprotein fragment and an HSV-1 glycoprotein or glycoprotein fragment. In another embodiment, the compositions used in the present invention and the methods of the present invention include a mixture of an HSV-2 glycoprotein or glycoprotein fragment and an HSV-1 glycoprotein or glycoprotein fragment. For example, in one embodiment, the composition of the present invention includes gC of HSV-2, gD of HSV-1, and gE of HSV-2, or fragments thereof. In another embodiment, the composition of the present invention includes gC of HSV-1, gD of HSV-2, and gE of HSV-2 or fragments thereof. In another embodiment, the composition of the present invention includes gC of HSV-2, gD of HSV-2, and gE of HSV-1, or fragments thereof. In another embodiment, the composition of the present invention includes gC of HSV-1, gD of HSV-1, and gE of HSV-2, or fragments thereof. In another embodiment, the composition of the present invention includes gC of HSV-1, gD of HSV-2, and gE of HSV-1, or fragments thereof. In another embodiment, the composition of the present invention includes gC of HSV-2, gD of HSV-1, and gE of HSV-1, or fragments thereof.

[0069] In another embodiment, the composition of the present invention includes one or more additional HSV-1 glycoproteins or HSV-2 glycoproteins or both HSV-1 and HSV-2 glycoproteins, as described herein. For example, in one embodiment, the composition of the present invention that includes gC of HSV-2, gD of HSV-1, and gE of HSV-2 may further include gI of HSV-1. In another embodiment, the composition of the present invention that includes gC of HSV-2, gD of HSV-2, and gE of HSV-2 may further include gB of HSV-1. Each of the possible combinations of HSV-1 and HSV-2 glycoproteins represents an individual embodiment of the present invention.

[0070] In one embodiment, "encoding" refers to an RNA molecule that contains a gene encoding a protein of interest. In another embodiment, the RNA molecule comprises a protein-coding sequence encoding the protein of interest. In another embodiment, one or more other proteins are also encoded. In another embodiment, the protein of interest is the only protein encoded. Each possibility represents an individual embodiment of the invention.

[0071] In another embodiment, "immunogenic fragment" refers to a portion of a protein that is immunogenic and that elicits a protective immune response when administered to a subject.

[0072] In one embodiment, "immunogenic" or "immunogenicity" herein refers to the innate ability of a protein, peptide, nucleic acid, antigen, or organism to elicit an immune response in an animal when administered to the animal. Thus, in one embodiment, "enhancing immunogenicity" refers to increasing the ability of a protein, peptide, nucleic acid, antigen, or organism to elicit an immune response in an animal when administered to the animal. The increase in the ability of a protein, peptide, nucleic acid, antigen, or organism to elicit an immune response can, in one embodiment, be measured by an increase in the number of antibodies against the protein, peptide, nucleic acid, antigen, or organism, an increase in the diversity of antibodies against the antigen or organism, an increase in the number of T cells specific for the protein, peptide, nucleic acid, antigen, or organism, an increase in the cytotoxic or helper T cell response against the protein, peptide, nucleic acid, antigen, or organism, etc.

[0073] In one embodiment, the immunogenic polypeptide is also antigenic. "Antigenic", in another embodiment, refers to a peptide capable of specifically interacting with an antigen recognition molecule of the immune system, such as, for example, an immunoglobulin (antibody) or a T cell antigen receptor. The antigenic peptide, in another embodiment, contains an epitope of at least about 8 amino acids (AA). The antigenic portion of the polypeptide, also referred to herein as an epitope in one embodiment, can be that portion which is immunodominant for antibody or T cell receptor recognition, or can be the portion used to generate an antibody against the molecule by conjugating the antigenic portion to a carrier polypeptide for immunization. A molecule that is antigenic need not itself be immunogenic, i.e., capable of inducing an immune response without a carrier.

[0074] In one embodiment, "function" within the meaning of this invention refers to the innate ability of a protein, peptide, nucleic acid, fragment or variant thereof to exhibit a biological activity or function. In one embodiment, such biological functions are, for example, its binding properties to an interaction partner, such as a membrane-bound receptor, and in another embodiment, its trimerization properties. In the case of the functional fragments and functional variants of this invention, these biological functions may actually vary, for example, depending on their specificity or selectivity, but the basic biological functions are retained.

[0075] In one embodiment, the term "fragment" is used herein to refer to a protein or polypeptide that is shorter than the full-length protein or polypeptide or contains fewer amino acids. In another embodiment, a fragment refers to a nucleic acid encoding a protein fragment that is shorter than the full-length nucleic acid or contains fewer nucleotides. In another embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment is an internal section within the sequence of a protein, peptide, or nucleic acid. In another embodiment, the fragment is an immunogenic internal section within the sequence of a protein, peptide, or nucleic acid. In another embodiment, the fragment is a functional internal section within the sequence of a protein, peptide, or nucleic acid. In another embodiment, the fragment is an N-terminal immunogenic fragment. In one embodiment, 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 contains a piece of a protein that binds together or pieces of multiple proteins that bind together.

[0076] Thus, in one embodiment, the "immunogenic fragment" of a protein described in the present invention is immunogenic and, in some embodiments and in other embodiments, refers to a portion of the protein that elicits a protective immune response when administered to a subject.

[0077] In another aspect, the present invention provides a composition comprising a modified mRNA, wherein each of the modified mRNAs encodes a) herpes simplex virus glycoprotein D (gD) or an immunogenic fragment thereof, b) herpes simplex virus glycoprotein C (gC) or an immunogenic fragment thereof, c) herpes simplex virus glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof.

[0078] In one embodiment, the present invention provides a composition comprising a modified mRNA encoding gD of HSV or an immunogenic fragment thereof, a modified mRNA encoding gC of HSV or an immunogenic fragment thereof, and a modified mRNA encoding gE of HSV or an immunogenic fragment thereof.

[0079] In one embodiment, the composition of the modified mRNA encoding gD-1 is protective against HSV-1 infection. Also, the combined composition of the modified mRNAs encoding gC-1 / gD-1 / gE-1 provides superior protection compared to a composition containing the modified mRNA encoding gC-1 alone, the modified mRNA encoding gD-1 alone, or the modified mRNA encoding gE-1 alone. Also, as provided herein, the composition of the modified mRNA encoding gD-2 is protective against HSV-2 infection (FIGS. 7 to 10). Also, the combined composition of the modified mRNAs encoding gC-2 / gD-2 / gE-2 provides superior protection compared to a composition containing the modified mRNA encoding gC-2 alone, the modified mRNA encoding gD-2 alone, or the modified mRNA encoding gE-2 alone.

[0080] In another embodiment, by including the modified mRNA encoding gC and / or the modified mRNA encoding gE in the composition of the present invention, the effect of the anti-gD antibody induced by the composition is increased. In another embodiment, by including the modified mRNA encoding gC and / or the modified mRNA encoding gE in the composition of the present invention, the dose of the modified mRNA encoding gD required to induce an antibody that inhibits the binding of gD to the cell receptor is increased. In another embodiment, by including the modified mRNA encoding gC and / or the modified mRNA encoding gE in the composition of the present invention, when the modified mRNA encoding gD is administered separately from the modified mRNA encoding the gC protein or the gE protein, the dose of the modified mRNA encoding gD required to induce an antibody that inhibits the binding of gD to the cell receptor is reduced.

[0081] In another embodiment, the composition of the present invention comprises a modified mRNA encoding gC and / or a modified mRNA encoding gE, thereby enhancing the effect of the innate immune response. In another embodiment, the innate immune response is an antibody-mediated immune response. In another embodiment, the innate immune response is a non-antibody-mediated immune response. In another embodiment, the innate immune response is an NK (natural killer) cell response. In another embodiment, the innate immune response is another innate immune response known in the art.

[0082] In another embodiment, by including a modified mRNA encoding gC and / or a modified mRNA encoding gE in the composition of the present invention, the effect of the antibody induced by the composition against one of the above glycoproteins is increased. In another embodiment, by including a modified mRNA encoding gC and / or a modified mRNA encoding gE in the composition of the present invention, when one of the glycoproteins is administered separately from one of the other glycoproteins, the dose of one of the above glycoproteins required to induce an antibody that inhibits the glycoprotein from binding to its cell receptor is reduced. Glycoprotein D

[0083] In one embodiment, the composition of the present invention comprises a modified mRNA encoding the gD protein of HSV-1. In another embodiment, the composition comprises a modified mRNA encoding a fragment of the gD protein of HSV-1.

[0084] In one embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of gD of HSV-1 comprises the following:

Chemical formula

Chemical formula

Chemical formula

[0085] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent the 5' untranslated sequence. In one embodiment, the bold residues represent a signal sequence (leader sequence) that aids in the expression of the gD1 fragment. In one embodiment, the italicized residues represent the 3' untranslated sequence and the polyadenylation tail.

[0086] In another embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of HSV-1 gD lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylation tail, or a combination thereof.

[0087] In one embodiment, the fragment of HSV-1 gD encoded by the modified mRNA used in the methods and compositions of the invention comprises amino acids 26 - 331 of gD from the Patton strain of HSV-1 as set forth in the following amino acid sequence: KYALADASLKMADPNRFRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPY (SEQ ID NO: 2)

[0088] In one embodiment, the full-length HSV-1 gD encoded by the modified mRNA used in the methods and compositions of the present invention comprises the following amino acid sequence: MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKLADPNRFRRKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEAPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (SEQ ID NO: 3)

[0089] In another embodiment, the HSV-1 gD encoded by the modified mRNA used in the methods and compositions of the present invention comprises the amino acid sequence specified in any one 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).

[0090] In another embodiment, the gD of HSV-1 encoded by the modified mRNA used in the methods and compositions of the invention comprises the amino acid sequence specified 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, ADM22466.1, ADM22542.1, ADM22619.1, ADM22696.1, ADM22773.1, ADM22849.1, ADM22926.1, ADM23003.1, ADM23079.1, ADM23155.1, ADM23231.1, ADM23309.1, ADM23383.1, ADM23457.1, ADM23531.1, ADM23605.1, ADM23680.1, ADM23755.1, ADM23831.1, AEQ77097.1, AER37647.1, AER37715.1, AER37786.1, 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, AJE60293.1, AJE60439.1, AKE48645.1, AKG59246.1, AKG59318.1, AKG59391.1, AKG59462.1, AKG59536.1, AKG59609.1, AKG59682.1, AKG59755.1, AKG59826.1, AKG59898.1, AKG59972.1, AKG60046.1, AKG60118.1, AKG60189.1, AKG60261.1, AKG60334.1, AKG60404.1, AKG60474.1, AKG60546.1, AKG60620.1, AKG60692.1, AKG60763.1, AKG60835.1. AKG60906.1, AKG60978.1, AKG61050.1, AKG61123.1, AKG61194.1, AKG61267.1, AKG61339.1, AKG61411.1, AKG61484.1, AKG61556.1, AKG61629.1, AKG61703.1, AKG61774.1, AKG61847.1, AKG61920.1, AKG61993.1, AKH80463.1, AKH80536.1, ALM22635.1, ALM22709.1, ALM22783.1, ALM22857.1, ALO18662.1, ALO18738.1, AMB65662.1, 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, ANN84806.1, ANN84883.1, ANN84959.1, ANN85036.1, ANN85112.1, ANN85187.1, ANN85264.1, ANN85341.1, ANN85416.1, ANN85494.1, ANN85571.1, ANN85648.1, ANN85724.1, ANN85801.1, AOY34093.1, AOY34141.1, AOY34243.1, AOY34271.1, AOY34337.1, AOY36685.1, ARB08957.1, AR037961.1, 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, ARO37980.1, 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, ASM47818.1, 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, SBS69579.1, SBS69625.1, SBS69688.1, SBS69694.1, SBS69717.1, SBS69727.1, SBS69811.1, SBT69395.1, SCL76902.1, VGBEDZ, or YP_009137141.1.

[0091] In another embodiment, the composition comprises a modified mRNA encoding the gD protein of HSV-2. In another embodiment, the composition comprises a modified mRNA encoding a fragment of the gD protein of HSV-2.

[0092] In one embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of gD of HSV-2 comprises the following:

Chemical formula

Chemical formula

[0093] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent the 5' untranslated region. In one embodiment, the bold residues represent a signal sequence (leader sequence) that aids in the expression of the gD2 fragment. In one embodiment, the italicized residues represent the 3' untranslated region and the polyadenylation tail.

[0094] In another embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of HSV-2 gD lacks a 5' untranslated region, a signal sequence, a 3' untranslated region, a polyadenylation tail, or a combination thereof.

[0095] In one embodiment, the fragment of HSV-2 gD encoded by the modified mRNA used in the methods and compositions of the present invention is HSV-2 as specified in the following amino acid sequence comprising amino acids 26-331 of gD from strain 333: KYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHH (SEQ ID NO: 5).

[0096] In one embodiment, the full-length HSV-2 gD encoded by the modified mRNA used in the methods and compositions of the present invention comprises the following amino acid sequence: MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (SEQ ID NO: 6).

[0097] In another embodiment, the gD of HSV-2 encoded by the modified mRNA used in the methods and compositions of the present invention comprises the amino acid sequences specified in the following GenBank accession numbers: 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, ABU45462.1, 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, AQZ56109.1, 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, AR038020.1, 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.

[0098] In another embodiment, the gD protein or fragment comprises Y63. In another embodiment, the gD protein or fragment comprises R159. In another embodiment, the gD protein or fragment comprises D240. In another embodiment, the gD protein or fragment comprises P246. In another embodiment, the gD protein or fragment comprises a residue selected from Y63, R159, D240, and P246. In another embodiment, inclusion of one of these residues elicits an antibody that inhibits binding to nectin-1.

[0099] The names used herein with respect to gD amino acid residues include the residues of the signal sequence. Thus, those of the mature protein residues are designated "26".

[0100] Each modified mRNA encoding the gD-1 and gD-2 proteins or fragments thereof represents an individual embodiment of the invention.

[0101] In another embodiment, the HSV gD, gC, and gE proteins and fragments thereof encoded by the modified mRNAs disclosed herein are described in U.S. Patent Publication No. 2013-0028925-A1, which is hereby incorporated by reference in its entirety.

[0102] In another embodiment, the fragment of the gD protein encoded by the modified mRNA used in the methods and compositions of the invention is an immunogenic fragment. In another embodiment, the gD immunoprotective antigen need not be the entire protein. In another embodiment, the protective immune response generally involves an antibody response. In another embodiment, variants of gD, sequence-conserved variants, and function-conserved variants are useful in the methods and compositions of the invention, provided that all such variants retain the required immunoprotective effect. In another embodiment, the immunogenic fragment can include an immunoprotective gD antigen from any strain of HSV. In another embodiment, the immunogenic fragment can include an HSV sequence variant as found in an infected individual. Glycoprotein C

[0103] In another embodiment, the composition of the invention comprises a modified mRNA encoding the gC protein of HSV-1. In another embodiment, the composition comprises a modified mRNA encoding a fragment of the gC protein of HSV-1.

[0104] In one embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of gC of HSV-1 comprises the following:

Chemical formula

Chemical formula

Chemical formula

[0105] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent the 5' untranslated sequence. In one embodiment, the bold residues represent a signal sequence (leader sequence) that aids in the expression of the gC1 fragment. In one embodiment, the italicized residues represent the 3' untranslated sequence and the polyadenylation tail.

[0106] In another embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of HSV-1 gC does not include a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylation tail, or a combination thereof.

[0107] In one embodiment, the fragment of HSV-1 gC encoded by the modified mRNA used in the methods and compositions of the present invention is HSV-1 as specified in the following amino acid sequence comprising amino acids 27 - 457 of gC from the KOS strain of HSV-1: ETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMYYLAWGRMDSPHEYGTWVRVRMFRPPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHH (SEQ ID NO: 8).

[0108] In one embodiment, the fragment of gC encoded by the modified mRNA used in the methods and compositions of the present invention comprises amino acids 27 - 457 of gC from an HSV-1 strain.

[0109] In one embodiment, the full-length HSV-1 gC encoded by the modified mRNA used in the methods and compositions of the present invention comprises the following amino acid sequence: MAPGRVGLAVVLWGLLWLGAGVAGGSETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMYYLAWGRMDSPHEYGTWVRVRMFRPPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHHGSHQPPPRDPTERQVIEAIEWVGIGIGVLAAGVLVVTAIVYVVRTSQSRQRHRR (SEQ ID NO: 9).

[0110] In another embodiment, the HSV-1 gC encoded by the modified mRNA used in the methods and compositions of the present invention comprises the amino acid sequence specified 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, ADM23057.1, 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, AKE98429.1, AKE98430.1, AKE98431.1, AKE98432.1, AKE98433.1, AKE98434.1, AKE98435.1, AKG59227.1, AKG59299.1, AKG59372.1, AKG59444.1, AKG59516.1, AKG59591.1, AKG59663.1, AKG59736.1, AKG59807.1, AKG59879.1, AKG59953.1, 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, AKG61175.1, AKG61248.1, AKG61321.1, AKG61392.1, AKG61464.1, AKG61537.1, AKG61611.1, AKG61684.1, AKG61756.1, AKG61828.1, AKG61902.1, AKG61974.1, AKH80444.1, AKH80517.1, AKM76368.1, ALM22613.1, ALM22687.1, ALM22761.1, ALM22835.1, ALO18641.1, ALO18717.1, AMB65642.1, AMB65715.1, AMB65862.1, AMN09813.1, ANN83942.1, ANN84019.1, ANN84095.1, ANN84172.1, ANN84249.1, ANN84326.1, ANN84403.1, ANN84478.1, ANN84555.1, ANN84632.1, ANN84708.1, ANN84785.1, ANN84861.1, ANN84938.1, ANN85014.1, ANN85091.1, 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, ARO38064.1, ARO38065.1, ARO38066.1, ASM47642.1, ASM47719.1, ASM47796.1, ASM47871.1, BAM73394.1, CAA32294.1, CAB40083.1, CAD13356.1, CAD13357.1, CAD13358.1, CAD13359.1, CAD13360.1, CAD13361.1, CAD13362.1, CAD13363.1, CAD13364.1, CAD13365.1, CAD13366.1, CAD13367.1, CAD13368.1, CAD13369.1, CAD13370.1, CAD13371.1, CAD13372.1, CAD13373.1, CAD13374.1, CAD13375.1, CAD13376.1, CAD13377.1, CAD13378.1, 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.

[0111] In another embodiment, the composition comprises a modified mRNA encoding the gC protein of HSV-2. In another embodiment, the composition comprises a modified mRNA encoding a fragment of the gC protein of HSV-2.

[0112] In one embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of gC of HSV-2 comprises the following:

Chemical formula

[0113] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent the 5'untranslated sequence. In one embodiment, the bold residues represent a signal sequence (leader sequence) that aids in the expression of the gC2 fragment. In one embodiment, the italicized residues represent the 3'untranslated sequence and the polyadenylation tail.

[0114] In another embodiment, the nucleotide sequence of the modified mRNA encoding the fragment of HSV-2 gC lacks a 5'untranslated sequence, a signal sequence, a 3'untranslated sequence, a polyadenylation tail, or a combination thereof.

[0115] In one embodiment, the fragment of HSV-2 gC encoded by the modified mRNA used in the methods and compositions of the invention is HSV-2 as specified in the following amino acid sequence Comprising amino acids 27 to 426 of gC from 333 strains: ASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTESRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH (SEQ ID NO: 11).

[0116] In one embodiment, the full-length HSV-2 gC encoded by the modified mRNA used in the methods and compositions of the invention comprises the following amino acid sequence: MALGRVGLAVGLWGLLWVGVVVVLANASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHHGSHQPPPRDPTERQVIRAVEGAGIGVAVLVAVVLAGTAVVYLTHASSVRYRRLR (SEQ ID NO: 12).

[0117] In another embodiment, the HSV-2 gC encoded by the modified mRNA used in the methods and compositions of the present invention comprises the amino acid sequence specified 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, AKC59285.1, 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, AQZ55948.1, AQZ56019.1, AQZ56090.1, AQZ56161.2, AQZ56232.2, AQZ56303.2, AQZ56374.2, AQZ56445.1, AQZ56516.1, AQZ56587.1, AQZ56658.1, AQZ56729.2, AQZ56800.1, AQZ56871.1, AQZ56942.2, AQZ57013.1, AQZ57084.2, AQZ57155.1, AQZ57226.1, AQZ57297.1, AQZ57368.1, AQZ57439.1, AQZ57510.1, AQZ57581.1, AQZ57652.1, AQZ57723.1, AQZ57794.2, AQZ57865.2, AQZ57936.1, AQZ58007.2, AQZ58078.1, AQZ58149.2, AQZ58220.1, AQZ58291.1, AQZ58362.1, AQZ58433.1, AQZ58504.1, AQZ58575.1, AQZ58646.1, AQZ58717.2, AQZ58788.2, AQZ58859.2, AQZ58930.1, AQZ59001.2, AQZ59072.1, AQZ59143.1, ARO38067.1, ARO38068.1. ARO38069.1, ARO38070.1, ARO38071.1, ARO38072.1, CAA25687.1, CAA26025.1, CAB06730.1, CAB06734.1, CAB96544.1, P03173.1, P06475.1, P89475.1, Q89730.1, YP_009137161.1, YP_009137196.1, or YP_009137220.1.

[0118] In another embodiment, the fragment of the gC protein encoded by the modified mRNA used in the methods and compositions of the present invention is, in one embodiment, a properdin-interfering domain that refers to a domain that blocks or inhibits the binding of a host C3b molecule to a host properdin molecule. In another embodiment, this term refers to a domain that blocks or inhibits the interaction of a host C3b molecule with a host properdin molecule.

[0119] In another embodiment, the fragment of the gC protein encoded by the modified mRNA used in the methods and compositions of the present invention is a C5 interfering domain. In another embodiment, the fragment of the gC protein is a portion of the C5 interfering domain. In another embodiment, the "C5 interfering domain" refers to a domain that interferes with the binding of a host C3b molecule to a host C5 molecule. In another embodiment, this term refers to a domain that interferes with the interaction of a host C3b molecule with a host C5 molecule.

[0120] Each modified mRNA encoding a gC-1 or gC-2 protein or a fragment thereof represents an individual embodiment of the present invention.

[0121] In another embodiment, the fragment of the gC protein encoded by the modified mRNA used in the methods and compositions of the invention is an immunogenic fragment. In another embodiment, the gC immunoprotective antigen need not be the entire protein. In another embodiment, the protective immune response generally involves an antibody response. In another embodiment, variants, sequence-conserved variants, and function-conserved variants of gC are useful in the methods and compositions of the invention, provided that all such variants retain the required immunoprotective effect. In another embodiment, the immunogenic fragment can comprise an immunoprotective gC antigen from any strain of HSV. In another embodiment, the immunogenic fragment can comprise an HSV sequence variant as seen in an infected individual. Glycoprotein E

[0122] In another embodiment, the composition of the invention comprises a modified mRNA encoding the gE protein of HSV-1. In another embodiment, the composition comprises a modified mRNA encoding a fragment of the gE protein of HSV-1.

[0123] In one embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of gD of HSV-1 comprises:

Chemical formula

Chemical formula

Chemical formula

[0124] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent the 5' untranslated sequence. In one embodiment, the bold residues represent a signal sequence (leader sequence) that aids in the expression of the gE1 fragment. In one embodiment, the italicized residues represent the 3' untranslated sequence and the polyadenylation tail.

[0125] In another embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of HSV-1 gE lacks a 5' untranslated sequence, a signal sequence, a 3' untranslated sequence, a polyadenylation tail, or a combination thereof.

[0126] In one embodiment, the fragment of HSV-1 gE encoded by the modified mRNA used in the methods and compositions of the invention is HSV-1 as set forth in the following amino acid sequence comprising amino acids 24 - 409 of gE from the NS strain: KTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGA (SEQ ID NO: 14).

[0127] In one embodiment, the fragment of gE encoded by the modified mRNA used in the methods and compositions of the invention comprises amino acids 24 - 409 of gE from an HSV-1 strain.

[0128] In one embodiment, the full-length HSV-1 gE encoded by the modified mRNA used in the methods and compositions of the invention comprises the following amino acid sequence: MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWRAVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPSTNGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW (SEQ ID NO: 15).

[0129] In another embodiment, the HSV-1 gE encoded by the modified mRNA used in the methods and compositions of the present invention comprises the amino acid sequence specified 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, ADM23311.1, 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, AKE98388.1, AKE98389.1, AKE98390.1, AKE98391.1, AKE98392.1, AKE98393.1, AKG59248.1, AKG59320.1, AKG59393.1, AKG59464.1, AKG59538.1, AKG59611.1, AKG59684.1, AKG59757.1, AKG59828.1, AKG59900.1, AKG59974.1, AKG60048.1, 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, AKG61269.1, AKG61341.1, AKG61413.1, AKG61486.1, AKG61558.1, AKG61631.1, AKG61705.1, AKG61776.1, AKG61849.1, AKG61922.1, AKG61995.1, AKH80465.1, AKH80538.1, ALM22637.1, ALM22711.1, ALM22785.1, ALM22859.1, ALO18664.1, 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, ANN84655.1, ANN84732.1, ANN84808.1, ANN84885.1, ANN84961.1, ANN85038.1, ANN85114.1, 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, ARO38079.1, ARO38080.1, ASM47642.1, ASM47666.1, ASM47743.1, ASM47820.1, ASM47895.1, BAM73421.1, CAA26062.1, CAA32272.1, CAF24756.1, CAF24757.1, CAF24758.1, CAF24759.1, CAF24760.1, CAF24761.1, CAF24762.1, CAF24763.1, CAF24764.1, CAF24765.1, CAF24766.1, CAF24767.1, CAF24768.1, CAF24769.1, CAF24770.1, 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, SBS69636.1, SBS69693.1, SBS69701.1, SBS69722.1, SBS69732.1, SBS69813.1, SBT69397.1, or YP_009137143.1.

[0130] In another embodiment, the composition comprises a modified mRNA encoding the gE protein of HSV-2. In another embodiment, the composition comprises a modified mRNA encoding a fragment of the gE protein of HSV-2.

[0131] In one embodiment, the nucleotide sequence of the modified mRNA encoding a fragment of gE of HSV-2 comprises the following:

Chemical formula

[0132] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent the 5' untranslated region. In one embodiment, the bold residues represent a signal sequence (leader sequence) that aids in the expression of the gE2 fragment. In one embodiment, the italicized residues represent the 3' untranslated region and the polyadenylation tail.

[0133] In another embodiment, the nucleotide sequence of the modified mRNA encoding the fragment of gE of HSV-2 lacks a 5' untranslated region, a signal sequence, a 3' untranslated region, a polyadenylation tail, or a combination thereof.

[0134] In one embodiment, the fragment of gE of HSV-2 encoded by the modified mRNA used in the methods and compositions of the present invention comprises amino acids 24-405 of gE from HSV-2 strain 2.12 as set forth in the following amino acid sequence: RTSWKRVTSGEDVVLLPAPAGPEERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPVPTPTPDDYDEEDDAGVSERTPVSVPPPTPPRRPPVAPPTHPRVIPEVSHVRGVTVHMETPEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCAEMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRA (SEQ ID NO: 17).

[0135] In one embodiment, the full-length HSV-2 gE encoded by the modified mRNA used in the methods and compositions of the present invention comprises the following amino acid sequence: MARGAGLVFFVGVWVVSCLAAAPRTSWKRVTSGEDVVLLPAPAERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPVPTPTPDDYDEEDDAGVTNARRSAFPPQPPPRRPPVAPPTHPRVIPEVSHVRGVTVHMETLEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCADMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRAPHPAPSARGPLRLGAVLGAALLLAALGLSAWACMTCWRRRSWRAVKSRASATGPTYIRVADSELYADWSSDSEGERDGSLWQDPPERPDSPSTNGSGFEILSPTAPSVYPHSEGRKSRRPLTTFGSGSPGRRHSQASYPSVLW (SEQ ID NO: 18).

[0136] In another embodiment, the gE of HSV-2 encoded by the modified mRNA used in the methods and compositions of the present invention comprises the amino acid sequence specified 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, ABW83334.1, 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, AKC59307.1, AKC59378.1, AKC59449.1, AKC59520.1, AKC59591.1, AMB66104.1, AMB66173.1, AMB66246.1, AMB66465.1, AQZ55756.1,, AQZ55827.1, AQZ55898.1, AQZ55969.2, AQZ56040.2, AQZ56111.2, AQZ56182.1, AQZ56253.2, AQZ56324.1, AQZ56395.1, AQZ56466.2, AQZ56537.1, AQZ56608.1, AQZ56679.1, 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.

[0137] In another embodiment, the fragment of gE encoded by the modified mRNA used in the methods and compositions of the invention comprises the IgG Fc binding domain of the gE protein. In another embodiment, the gE domain encoded by the modified mRNA used in the methods and compositions of the invention is another gE domain known in the art that mediates binding to IgG Fc.

[0138] In another embodiment, the gE protein encoded by the modified mRNA used in the methods and compositions of the invention comprises a gE domain involved in cell-to-cell spread.

[0139] In another embodiment, the fragment of gE encoded by the modified mRNA fragment used in the methods and compositions of the invention comprises an immune evasion domain. In another embodiment, the fragment of gE encoded by the modified mRNA fragment used in the methods and compositions of the invention comprises a portion of the immune evasion domain.

[0140] Each modified mRNA encoding the protein gE-1 or gE-2 or a fragment thereof represents an individual embodiment of the present invention.

[0141] In another embodiment, the fragment of the gE protein encoded by the modified mRNA used in the methods and compositions of the present invention is an immunogenic fragment. In another embodiment, the gE immunoprotective antigen need not be the entire protein. In another embodiment, the protective immune response generally involves an antibody response. In another embodiment, variants, sequence-conserved variants, and function-conserved variants of gE are useful in the methods and compositions of the present invention, provided that all such variants retain the required immunoprotective effect. In another embodiment, the immunogenic fragment can comprise an immunoprotective gE antigen from any strain of HSV. In another embodiment, the immunogenic fragment can comprise a sequence variant of HSV as seen in an infected individual.

[0142] In one embodiment, the HSV glycoprotein encoded by the modified mRNA used in the methods and compositions of the present invention is a homolog of the sequences provided herein. In another embodiment, the HSV glycoprotein encoded by the modified mRNA used in the methods and compositions of the present invention is an isoform of the sequences provided herein. In another embodiment, the HSV glycoprotein encoded by the modified mRNA used in the methods and compositions of the present invention is a variant of the sequences provided herein. In another embodiment, the HSV glycoprotein encoded by the modified mRNA used in the methods and compositions of the present invention is a fragment of the sequences provided herein.

[0143] In another embodiment, the fragment of the glycoprotein encoded by the modified mRNA of the methods and compositions of the invention comprises the ectodomain of the glycoprotein. In another embodiment, the fragment of the glycoprotein encoded by the modified mRNA of the methods and compositions of the invention consists of the ectodomain of the glycoprotein. In another embodiment, the fragment of the glycoprotein encoded by the modified mRNA of the methods and compositions of the invention comprises a fragment of the ectodomain of the glycoprotein. In another embodiment, the fragment of the glycoprotein may be a fragment of any glycoprotein known in the art.

[0144] In another embodiment, the glycoprotein or immunogenic fragment encoded by the modified mRNA fragment used in the methods and compositions of the invention can be from any strain of HSV. In another embodiment, the immunogenic fragment encoded by the modified mRNA fragment used in the methods and compositions of the invention can include sequence variants of HSV as seen in infected individuals.

[0145] In one embodiment, a "variant" refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue), such as a splice variant, that is different from the majority of a parental population but is still sufficiently similar to be considered one of them. In one embodiment, the variant can be a sequence-conserved variant, while in another embodiment, the variant can be a function-conserved variant. In one embodiment, the variant can include the addition, deletion, or substitution of one or more amino acids.

[0146] In one embodiment, an "immune evasion 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-gD antibody). In another embodiment, this domain interferes with or reduces the in vivo anti-HSV efficacy of the anti-HSV immune response. In another embodiment, this domain reduces the immunogenicity of an HSV protein (e.g., gD) during a subsequent infection. In another embodiment, this domain reduces the immunogenicity of an HSV protein during a subsequent challenge. In another embodiment, this domain reduces the immunogenicity of HSV during a subsequent challenge. In another embodiment, this domain reduces the immunogenicity of an HSV protein in the context of an ongoing HSV infection. In another embodiment, this domain reduces the immunogenicity of HSV in the context of an ongoing HSV infection. In another embodiment, this domain functions as an Fc receptor for IgG. In another embodiment, this domain promotes the cross-linking of the bipolar of the antibody, which in one embodiment binds to the HSV antigen by its Fab domain and binds to a distant HSV antigen, e.g., gE in one embodiment, by its Fc domain, thereby blocking the ability of the Fc domain to activate complement. This is a term that refers to an antibody molecule that binds to an HSV antigen by its Fab domain and binds to a distant HSV antigen, e.g., gE in one embodiment, by its Fc domain, thereby blocking the ability of the Fc domain to activate complement.

[0147] The present invention also provides a modified mRNA encoding an analog of an HSV protein or polypeptide or a fragment thereof. The analog may differ from a naturally occurring protein or peptide by conservative amino acid sequence substitutions or by modifications that do not affect the sequence or by both.

[0148] In another embodiment, the HSV glycoprotein encoded by the modified mRNA of the present invention is a homolog to the sequences specified above, either explicitly or by reference to GenBank entries. The terms "homologous", "homolog", etc. when referring to any protein or peptide, in one embodiment, refer to the percentage of amino acid residues in a candidate sequence that match the residues of the corresponding native polypeptide after aligning the sequences to introduce gaps and achieving the maximum percent homology if necessary, and any conservative substitutions are not considered part of the sequence identity. Methods of alignment and computer programs are well known in the art.

[0149] In another embodiment, "homology" refers to the identity of the protein sequence encoded by the modified mRNA that is greater than 70% to the sequences 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%.

[0150] In one embodiment, an "isoform" is a variant of a molecule, such as a protein, that is a variant of the same protein with only minor differences. In one embodiment, an isoform can be produced from different but related genes, or in another embodiment, can arise from the same gene by alternative splicing. In another embodiment, an isoform is caused by a polymorphism of a single nucleotide.

[0151] In another embodiment, the modified mRNA encoding the glycoprotein or a fragment of the glycoprotein described herein further encodes an antigenic tag. In one embodiment, this tag is a histidine ("His") tag. In one embodiment, the His tag contains five histidine residues. In another embodiment, the His tag contains six histidine residues.

[0152] In another embodiment, the methods and compositions of the invention utilize chimeric molecules, which comprise a fusion of a modified mRNA encoding a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind and a modified mRNA encoding an HSV protein. In other embodiments, the epitope tag is located at the amino or carboxy terminus of the protein, or at an internal location therein. The presence of such epitope-labeled forms of the recombinant HSV protein is, in another embodiment, detected by an antibody to the tag polypeptide. In another embodiment, the inclusion of an epitope tag enables the easy purification of the recombinant HSV protein by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. A variety of tag polypeptides and their respective antibodies are known in the art.

[0153] In one embodiment, the composition of the present invention contains an adjuvant, while in another embodiment, the composition does not contain an adjuvant. "Adjuvant" refers to, in another embodiment, a compound that increases the immune response to an antigen in an individual to whom the antigen is administered or in a test system when tested in vitro. In another embodiment, an immune adjuvant promotes the immune response to an antigen that is weakly immunogenic when administered alone, i.e., does not induce or induces a weak antibody titer or induces a cell-mediated immune response. In another embodiment, the adjuvant increases the antibody titer against the antigen. In another embodiment, the adjuvant reduces the dose of the antigen effective to achieve an immune response in an individual. Multiple types of adjuvants are known in the art and are described in detail in US Patent Publication No. 2013 / 0028925, which is hereby incorporated herein by reference. Modified mRNA

[0154] In one embodiment, the present invention provides a composition comprising modified mRNA and methods of using the same. In one embodiment, the modified mRNA comprises one or more modified nucleoside residues.

[0155] In another embodiment, the modified nucleoside of the method and composition of the present invention 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).

[0156] In other embodiments, the modified nucleoside is m 1 A (1-methyladenosine), m 2 A (2-methyladenosine), m 6A (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-isopentenyladenosine), io 6 A (N6-(cis-hydroxyisopentenyl)adenosine), ms 2 io 6 A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g 6 A (N6-glycinylcarbamoyladenosine), 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-hydroxynorvalylcarbamoyladenosine), ms 2 hn 6 A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine), Ar(p)(2’-O-ribosyladenosine(phosphate)), I (inosine), m 1 I (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 (lysidine), m 1 G (1-methylguanosine), m 2 G (N2-methylguanosine), m7 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 (wybutosine), o2yW (peroxywybutosine), OHyW (hydroxywybutosine), OHyW * (modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQ0 (7-cyano-7-deazaguanosine), preQ1 (7-aminomethyl-7-deazaguanosine), G + (archaeosine), Ψ (pseudouridine) , D (dihydrouridine), m 5 U (5-methyluridine), Um (2’-O-methyluridine), m 5 Um (5,2’-O-dimethyluridine), m 1 Ψ (1-methylpseudouridine), Ψm (2’-O-methylpseudouridine), 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), cmo 5 U (uridine 5-oxyacetic acid), mcmo 5 U (uridine 5-oxyacetic acid methyl ester), chm 5 U (5-(carboxyhydroxymethyl)uridine), mchm 5U(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 2,2,7G(N2,N2,7-trimethylguanosine), m 3 Um(3,2’-O-dimethyluridine), m 5 D(5-methyldihydrouridine), m 3 Ψ(3-methylpseudouridine), 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-taurinomethyluridine), τm 5 s 2 U(5-taurinomethyl-2-thiouridine), imG-14(4-demethylwyosine), imG2(isowyosine), ac 6 A(N 6-acetyladenosine), inm 5 U(5-(isopentenylaminomethyl)uridine), inm 5 s2U(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 + (agmatidine), 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 cnm 5 is U(5-cyanomethyl-uridine).

[0157] In one embodiment, the modified nucleoside residue is pseudouridine or a pseudouridine family residue.

[0158] In one embodiment, the modified mRNA contains pseudouridine residues. In one embodiment, pseudouridine refers to the C-glycoside isomer of the nucleoside uridine. In one embodiment, the pseudouridine residue is m 1 acp 3 Ψ(1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, m 1 Ψ(1-methylpseudouridine), Ψm(2'-O-methylpseudouridine, m 5 D(5-methyldihydrouridine), m 3 Ψ(3-methylpseudouridine), or a combination thereof. In one embodiment, the pseudouridine residue contains a 1-methylpseudouridine residue in place of uridine.

[0159] In one embodiment, the modified nucleoside residue is a pseudouridine analog. In one embodiment, a "pseudouridine analog" is any modification, variant, isoform, or derivative of pseudouridine. For example, pseudouridine analogs include 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine (m 1 Ψ), 1-methyl-4-thio-pseudouridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3including, but not limited to, pseudouridine (Ψ), and 2'-O-methyl-pseudouridine (Ψm).

[0160] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (Ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6- aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (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-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τcm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τrm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleobase deoxythymine), 1-methylpseudouridine (m 1 Ψ), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-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 (1-methylpseudouridine (m 1 Ψ) also known as, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-β-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-β-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine (2'-F-ara-uridine), 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0161] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine are 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 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-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrole-cytidine, pyrrole-pseudoisocytidine, 2-thio-cytidine (s 2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, 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-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (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-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0162] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine are 2-amino-purine, 2,6-diaminopurine, 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-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 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 (i6 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-Glycinylcarbamoyl-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-Hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 2-Methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-Acetyl-adenosine (ac 6A ), 7-Methyl-adenine, 2-Methylthio-adenine, 2-Methoxy-adenine, α-Thio-adenosine, 2’-O-Methyl-adenosine (Am), N6,2’-O-Dimethyl-adenosine (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-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-Amino-pentaoxanonadecyl)-adenosine.

[0163] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxwybutosine (o2yW), hydroxywybutosine (OHyW), modified hydroxywybutosine (OHyW * ), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (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 2’7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), and 2'-O-ribosyl guanosine (phosphate) (Gr(p)).

[0164] The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, purine or pyrimidine analogs. For example, the nucleobases can each be independently selected from adenine, cytosine, guanine, uracil or hypoxanthine. In another embodiment, the nucleobases can also be, for example, 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-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; and 1,3,5triazine, including natural and synthetic derivatives of the bases. When the nucleotides are represented using the abbreviated forms A, G, C, T or U, each letter refers to a representative base and / or its derivative, e.g., A includes adenine or an adenine analog, e.g., 7-deazaadenine). Modifications in internucleoside linkages

[0165] Modified nucleotides can be incorporated into polynucleotides, primary constructs, or mRNA molecules and can be modified at internucleoside linkages (e.g., the phosphate backbone). In the context of the polynucleotide backbone, the terms "phosphate" and "phosphodiester" are used interchangeably herein. The phosphate groups of the backbone can be modified by substituting one or more of the oxygen atoms with different substituents. Also, modified nucleosides and nucleotides can include large-scale substitution of unmodified phosphate moieties with other internucleoside linkages as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphor selenate, borano phosphate, borano phosphate ester, hydrogen phosphonate, phosphoramidate, phosphorodiamidate, alkyl or aryl phosphonate, and phosphotriester. Phosphorodithioate has both unbonded oxygens replaced by sulfur. The phosphate linker can also be modified by replacing the bonded oxygen with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene-phosphonate).

[0166] α-Thio-substituted phosphate moieties are provided to confer stability to RNA and DNA polymers via non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA are more nuclease-resistant and thus have a longer half-life in the cellular environment. Phosphorothioate-linked polynucleotides, primary constructs, or mmRNA molecules are also expected to reduce the innate immune response by weaker binding / activation of the cell's innate immune molecules.

[0167] In certain embodiments, the modified nucleoside comprises an alpha-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (alpha-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0168] Other internucleoside linkages that can be employed in accordance with the present invention, including internucleoside linkages that do not contain a phosphorus atom, are described hereinbelow. Combinations of modified sugars, nucleobases, and internucleoside linkages

[0169] The polynucleotides, primary constructs, and mmRNAs of this invention can include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkage.

[0170] In another embodiment, the purified formulations of the RNAs, oligoribonucleotides, or polyribonucleotides of the methods and compositions of the present invention include combinations of two or more of the above-described modifications. In another embodiment, the purified formulations of the RNAs or oligoribonucleotides include combinations of three or more of the above-described modifications. In another embodiment, the purified formulations of the RNAs or oligoribonucleotides include combinations of more than three of the above-described modifications.

[0171] In one embodiment, the modified mRNA includes modified mRNA synthesized in vitro.

[0172] In one embodiment, the present invention includes one or more modified mRNAs encoding HSV glycoproteins. In one embodiment, the modified RNA includes pseudouridine or a pseudouridine family residue. In another embodiment, the modified mRNA of the present invention is capable of controlling the protein expression of the HSV glycoprotein encoded thereby.

[0173] In another embodiment, the invention provides an in vitro transcribed mRNA molecule encoding an HSV glycoprotein, which contains pseudouridine. In another embodiment, the invention provides a synthetic mRNA molecule encoding an HSV glycoprotein, which contains pseudouridine.

[0174] In another embodiment, the in vitro transcribed mRNA molecule of the methods and compositions of the invention 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 polymerases described above. In another embodiment, the mRNA is chemically synthesized on a cylinder similar to DNA.

[0175] In another embodiment, the nucleoside modified in the RNA, oligoribonucleotide, or polynucleotide of the methods and compositions of the invention 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).

[0176] In another embodiment, the modified mRNA of the methods and compositions of the invention further comprises a poly-A tail. In another embodiment, the modified mRNA of the methods and compositions of the invention does not contain a poly-A tail. Each possibility represents an individual embodiment of the invention.

[0177] In another embodiment, the modified mRNA of the methods and compositions of the present invention comprises an m7GpppG cap. In another embodiment, the modified mRNA of the methods and compositions of the present invention does not comprise an m7GpppG cap. In another embodiment, the modified mRNA of the methods and compositions of the present invention comprises 3'-O-methyl-m7GpppG. In another embodiment, the modified mRNA of the methods and compositions of the present invention comprises an irreversible cap analog, which in one embodiment is added during mRNA transcription. In another embodiment, the modified mRNA of the methods and compositions of the present invention comprises an anti-reverse cap analog. Each possibility represents an individual embodiment of the present invention.

[0178] In another embodiment, the modified mRNA of the methods and compositions of the present invention further comprises a cap-independent translation enhancer. In another embodiment, the modified mRNA of the methods and compositions of the present invention does not comprise a cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is the cap-independent translation enhancer of tobacco etch virus (TEV). In another embodiment, the cap-independent translation enhancer is other cap-independent translation enhancers known in the art. Each possibility represents an individual embodiment of the present invention.

[0179] In one embodiment, "pseudouridine" is 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 3Refers to Ψ(3-methylpseudouridine). In another embodiment, the modified nucleoside is 4'(pseudouridine). In another embodiment, the term refers to an unmodified pseudouridine moiety. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the above-described pseudouridines. In another embodiment, the term refers to other pseudouridines known in the art. Each possibility represents an individual embodiment of the present invention.

[0180] In another embodiment, the modified RNA contains a modified nucleoside, which in one embodiment is m 5 C, m5U, m 6 A, s 2 U, Ψ, 2'-O-methyl-U, 2'-O-methylpseudouridine, or a combination thereof.

[0181] In another embodiment, the present invention provides a method for delivering a recombinant protein to a subject, the method comprising contacting the subject with the modified mRNA of the methods and compositions of the present invention thereby delivering the recombinant protein to the subject.

[0182] In another embodiment, the method of the present invention includes increasing the number, proportion or frequency of modified uridine nucleosides in an RNA molecule to reduce immunogenicity or increase the efficiency of translation. In one embodiment, the number of modified uridine residues in an RNA, oligoribonucleotide or polynucleotide molecule determines the magnitude of the effect observed in the present invention.

[0183] In another embodiment, 0.1% to 100% of the uridine residues in the modified mRNA of the methods and compositions of the invention are modified (e.g., by the presence of pseudouridine). In another embodiment, 0.1% of the residues are modified. In another embodiment, it is 0.2%. In another embodiment, the proportion is 0.3%. In another embodiment, the proportion is 0.4%. In another embodiment, the proportion is 0.5%. In another embodiment, the proportion is 0.6%. In another embodiment, the proportion is 0.8%. In another embodiment, the proportion is 1%. In another embodiment, the proportion is 1.5%. In another embodiment, the proportion is 2%. In another embodiment, the proportion is 2.5%. In another embodiment, the proportion is 3%. In another embodiment, the proportion is 4%. In another embodiment, the proportion is 5%. In another embodiment, the proportion is 6%. In another embodiment, the proportion is 8%. In another embodiment, the proportion is 10%. In another embodiment, the proportion is 12%. In another embodiment, the proportion is 14%. In another embodiment, the proportion is 16%. In another embodiment, the proportion is 18%. In another embodiment, the proportion is 20%. In another embodiment, the proportion is 25%. In another embodiment, the proportion is 30%. In another embodiment, the proportion is 35%. In another embodiment, the proportion is 40%. In another embodiment, the proportion is 45%. In another embodiment, the proportion is 50%. In another embodiment, the proportion is 60%. In another embodiment, the proportion is 70%. In another embodiment, the proportion is 80%. In another embodiment, the proportion is 90%. In another embodiment, the proportion is 100%.

[0184] In another embodiment, the ratio is less than 5%. In another embodiment, the ratio is less than 3%. In another embodiment, the ratio is less than 1%. In another embodiment, the ratio is less than 2%. In another embodiment, the ratio is less than 4%. In another embodiment, the ratio is less than 6%. In another embodiment, the ratio is less than 8%. In another embodiment, the ratio is less than 10%. In another embodiment, the ratio is less than 12%. In another embodiment, the ratio is less than 15%. In another embodiment, the ratio is less than 20%. In another embodiment, the ratio is less than 30%. In another embodiment, the ratio is less than 40%. In another embodiment, the ratio is less than 50%. In another embodiment, the ratio is less than 60%. In another embodiment, the ratio is less than 70%.

[0185] In another embodiment, 0.1% of the residues of a given uridine nucleotide are modified. In another embodiment, the proportion of nucleotides is 0.2%. In another embodiment, the proportion is 0.3%. In another embodiment, the proportion is 0.4%. In another embodiment, the proportion is 0.5%. In another embodiment, the proportion is 0.6%. In another embodiment, the proportion is 0.8%. In another embodiment, the proportion is 1%. In another embodiment, the proportion is 1.5%. In another embodiment, the proportion is 2%. In another embodiment, the proportion is 2.5%. In another embodiment, the proportion is 3%. In another embodiment, the proportion is 4%. In another embodiment, the proportion is 5%. In another embodiment, the proportion is 6%. In another embodiment, the proportion is 8%. In another embodiment, the proportion is 10%. In another embodiment, the proportion is 12%. In another embodiment, the proportion is 14%. In another embodiment, the proportion is 16%. In another embodiment, the proportion is 18%. In another embodiment, the proportion is 20%. In another embodiment, the proportion is 25%. In another embodiment, the proportion is 30%. In another embodiment, the proportion is 35%. In another embodiment, the proportion is 40%. In another embodiment, the proportion is 45%. In another embodiment, the proportion is 50%. In another embodiment, the proportion is 60%. In another embodiment, the proportion is 70%. In another embodiment, the proportion is 80%. In another embodiment, the proportion is 90%. In another embodiment, the proportion is 100%.

[0186] In another embodiment, the percentage of a given uridine nucleotide is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0187] In another embodiment, the terms "ribonucleotide", "oligoribonucleotide" and polynucleotide refer, in one embodiment, to a compound containing a nucleotide in which the sugar moiety is ribose. In another embodiment, the term includes both RNAs and RNA derivatives in which the backbone is modified. Since various RNA backbone modifications are known in the art, they are contemplated in the present invention. In one embodiment, the modified RNA is PNA (peptide nucleic acid). PNA containing a peptide backbone and nucleotide bases can, in another embodiment, bind to both DNA and RNA molecules. In another embodiment, the nucleotide is modified by substitution of one or more phosphodiester bonds with phosphorothioate bonds. In another embodiment, the artificial nucleic acid contains other variants of the phosphate backbone of natural nucleic acids known in the art. Derivatives of each nucleic acid represent individual embodiments of the present invention.

[0188] Methods for producing nucleic acids with modified backbones are well known in the art and are described, for example, in U.S. Pat. Nos. 5,723,335 and 5,663,153 issued to Hutcherson et al. and in PCT Publication WO 95 / 26204, which is related. Each method represents an individual embodiment of the invention.

[0189] The nucleic acid of interest can be purified by any method known in the art or any method to be developed, provided that the method removes contaminants from the nucleic acid preparation, thereby substantially reducing the immunogenic performance of the nucleic acid preparation. In one embodiment, the nucleic acid of interest is purified by 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 nucleic acid purification method 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 the present invention are separation by liquid chromatography and enzymatic digestion, each used alone or in any combination, simultaneously or in any order. Non-limiting examples of separation by liquid chromatography include HPLC and fast protein liquid chromatography (FPLC). Materials useful in the HPLC and FPLC methods of the present invention are cross-linked polystyrene / divinylbenzene (PS / DVB), PS / DVB-C18, PS / DVB-alkylated, Helix DNA columns (Varian), Eclipse dsDNA Analysis columns (Agilent Technologies Technologies), reverse phase 5 (RPC-5) exchange materials, DNAPac, ProSwift, and bio-inert UltiMate® 3000 titanium columns (Dionex), among others. Enzymes useful in the enzymatic digestion method of the present invention include any enzyme capable of digesting any contaminant in the nucleic acid preparation of this invention, such as, for example, dsRNA contaminants, and also include RNase III, RNase VI, Dicer, and Chipper (see Fruscoloni et al., 2002, PNAS 100:1639) including, but not limited to. Non-limiting examples of assays for assessing the purity of the nucleic acid of interest include dot blot assays, Northern blot assays, and dendritic cell activation assays, described elsewhere herein.

[0190] In another embodiment, the modified mRNA of the methods and compositions of the present invention is significantly less immunogenic than an unmodified in vitro synthesized mRNA molecule having the same sequence. In another embodiment, the modified mRNA molecule is two-fold less immunogenic than its unmodified equivalent. In another embodiment, the immunogenicity is reduced three-fold. In another embodiment, the immunogenicity is reduced five-fold. In another embodiment, the immunogenicity is reduced seven-fold. In another embodiment, the immunogenicity is reduced ten-fold. In another embodiment, the immunogenicity is reduced fifteen-fold. In another embodiment, the immunogenicity is reduced by a certain factor. In another embodiment, the immunogenicity is reduced fifty-fold. In another embodiment, the immunogenicity is reduced one hundred-fold. In another embodiment, the immunogenicity is reduced two hundred-fold. In another embodiment, the immunogenicity is reduced five hundred-fold. In another embodiment, the immunogenicity is reduced one thousand-fold. In another embodiment, the immunogenicity is reduced two thousand-fold. In another embodiment, the immunogenicity is reduced by a different factor difference.

[0191] In another embodiment, "significantly lower immunogenicity" refers to a detectable decrease in immunogenicity. In another embodiment, the term refers to a decrease by a certain multiple of immunogenicity (e.g., one of the multiples of decrease listed above). In another embodiment, the term refers to a decrease such that it is possible to administer an effective amount of the modified mRNA without inducing a detectable immune response. In another embodiment, the term refers to a decrease such that it is possible to repeatedly administer the modified mRNA without inducing an immune response sufficient to detectably reduce the expression of the recombinant protein. In another embodiment, it is a decrease such that it is possible to repeatedly administer the modified mRNA without inducing an immune response sufficient to induce a detectable expression of the recombinant protein.

[0192] Methods for determining immunogenicity are well known in the art and are described in detail in U.S. Patent No. 8,278,036, which is hereby incorporated by reference herein.

[0193] In another embodiment, the modified mRNA of the methods and compositions of the present invention is translated more efficiently intracellularly than an unmodified mRNA molecule having the same sequence. In another embodiment, the modified mRNA exhibits facilitation of the ability to be translated by target cells. In another embodiment, translation is facilitated 2-fold compared to its unmodified equivalent. In another embodiment, translation is facilitated 3-fold. In another embodiment, translation is facilitated 5-fold. In another embodiment, translation is facilitated 7-fold. In another embodiment, translation is facilitated 10-fold. In another embodiment, translation is facilitated 15-fold. In another embodiment, translation is facilitated 20-fold. In another embodiment, translation is facilitated 50-fold. In another embodiment, translation is facilitated 100-fold. In another embodiment, translation is facilitated 200-fold. In another embodiment, translation is facilitated 500-fold. In another embodiment, translation is facilitated 1000-fold. In another embodiment, translation is facilitated 2000-fold. In another embodiment, the rate is 10 to 1000-fold. In another embodiment, the rate is 10 to 100-fold. In another embodiment, the rate is 10 to 200-fold. In another embodiment, the rate is 10 to 300-fold. In another embodiment, the rate is 10 to 500-fold. In another embodiment, the rate is 20 to 1000-fold. In another embodiment, the rate is 30 to 1000-fold. In another embodiment, the rate is 50 to 1000-fold. In another embodiment, the rate is 100 to 1000-fold. In another embodiment, the rate is 200 to 1000-fold. In another embodiment, translation is facilitated by other significant amounts or ranges of amounts. Each possibility represents an individual embodiment of the present invention.

[0194] Methods for determining translation efficiency are well known in the art and include, for example, measuring the activity of a coded reporter protein (e.g., luciferase or jellyfish or green fluorescent protein [Wall A A, Phillips A M 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. J (Ngosuwan J, Wang N M et al, Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of pre-secretory proteins into the endoplasmic reticulum. J Biol Chem 2005; 280(30): 27670-8]), or measuring a radiolabel incorporated into the translated protein (Ngosuwan J, Wang N M et al, Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of pre-secretory proteins into the endoplasmic reticulum. J Biol Chem 2003; 278(9): 7034-42). Each method represents an individual embodiment of the invention.

[0195] In another embodiment, the labeled cells of the method of the invention are dendritic cells. In another embodiment, the labeled cells of the method of the invention are macrophages. In another embodiment, the labeled cells of the method of the invention are B cells. In another embodiment, the labeled cells of the method of the invention are another antigen-presenting cell. In another embodiment, the labeled cells of the method of the invention are mucosal cells. In another embodiment, the labeled cells of the method of the invention are epithelial cells. In another embodiment, the cells are skin cells. In another embodiment, the cells are epithelial cells. In another embodiment, the cells are keratinocytes. In another embodiment, the cells are Merkel cells, melanocytes or Langerhans cells. Each method represents an individual embodiment of the invention.

[0196] Methods of treatment and methods of using the compositions The invention also provides a method of vaccinating a subject against HSV and treating, interfering with, inhibiting, reducing the occurrence of, or suppressing HSV infection or its symptoms or signs, comprising the step of administering a composition of the invention.

[0197] In one embodiment, the present invention provides a method for treating HSV infection in a subject, comprising contacting the subject with a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof.

[0198] In another embodiment, the present invention provides a method for suppressing HSV infection in a subject, comprising contacting the subject with a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof.

[0199] In another embodiment, the present invention provides a method for inhibiting HSV infection in a subject, comprising contacting the subject with a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof.

[0200] In another embodiment, the present invention provides a method for reducing the occurrence of HSV infection in a subject, comprising contacting the subject with a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV glycoprotein or an immunogenic fragment thereof.

[0201] In one embodiment, the HSV infection is HSV-1 infection. In another embodiment, the HSV infection is HSV-2 infection.

[0202] In one embodiment, a subject is administered an HSV-1 glycoprotein for a method of treating, inhibiting, suppressing, etc. HSV-1 infection. In another embodiment, a subject is administered an HSV-2 glycoprotein for a method of treating, inhibiting, suppressing, etc. HSV-2 infection. In another embodiment, a subject is administered an HSV-1 glycoprotein for a method of treating, inhibiting, suppressing, etc. HSV-1 infection, HSV-2 infection, or a combination thereof. In another embodiment, a subject is administered an HSV-2 glycoprotein for a method of treating, inhibiting, suppressing, etc. HSV-1 infection, HSV-2 infection, or a combination thereof. In one embodiment, administration of an HSV-1 glycoprotein (e.g., gC1, gD1, gE1, or a combination thereof) treats or prevents HSV-1 and HSV-2 infections. In another embodiment, administration of an HSV-2 glycoprotein (e.g., gC2, gD2, and gE2, or a combination thereof) treats or prevents HSV-1 and HSV-2 infections.

[0203] In one aspect, in one embodiment, the invention provides a method of treating, suppressing, inhibiting, or reducing the occurrence of herpes simplex virus 1 (HSV-1) infection in a subject, the method comprising contacting the subject with a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV-1 glycoprotein or an immunogenic fragment thereof.

[0204] In one embodiment, the invention provides a method of treating, suppressing, inhibiting, or reducing the occurrence of herpes simplex virus 2 (HSV-2) infection in a subject, the method comprising contacting the subject with a composition comprising one or more modified mRNAs, each of the modified mRNAs encoding an HSV-2 glycoprotein or an immunogenic fragment thereof.

[0205] In one embodiment, the contacting step is via administration to the subject.

[0206] In another embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV infection in a subject, the method comprising administering to the subject an immunogenic composition comprising (a) HSV gD or an immunogenic fragment thereof; (b) HSV gC or a fragment thereof as described herein; (c) HSV gE or a fragment thereof as described herein, or a modified mRNA encoding a combination thereof.

[0207] In another embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV-2 infection in a subject, the method comprising administering to the subject an immunogenic composition comprising (a) HSV-2 gD or an immunogenic fragment thereof; (b) HSV-2 gC or a fragment thereof as described herein; (c) HSV-2 gE or a fragment thereof as described herein, or a modified mRNA encoding a combination thereof.

[0208] In another embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV-1 infection in a subject, the method comprising administering to the subject an immunogenic composition comprising (a) HSV-1 gD or an immunogenic fragment thereof; (b) HSV-1 gC or a fragment thereof as described herein; (c) HSV-1 gE or a fragment thereof as described herein, or a modified mRNA encoding a combination thereof.

[0209] In another embodiment, the present invention provides a method for inducing an anti-HSV immune response in a subject, the method comprising administering to the subject an immunogenic composition comprising (a) HSV gD or an immunogenic fragment thereof; (b) HSV gC or a fragment thereof as described herein; (c) HSV gE or a fragment thereof as described herein, or a modified mRNA encoding a combination thereof.

[0210] In another embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV infection in a subject, the method comprising administering to the subject an immunogenic composition comprising (a) HSV-2 gD or an immunogenic fragment thereof; (b) HSV-2 gC or a fragment thereof as described herein; (c) HSV-2 gE or a fragment thereof as described herein, or a modified mRNA encoding a combination thereof.

[0211] In another embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV infection in a subject, the method comprising administering to the subject an immunogenic composition comprising (a) HSV-1 gD or an immunogenic fragment thereof; (b) HSV-1 gC or a fragment thereof as described herein; (c) HSV-1 gE or a fragment thereof as described herein, or a modified mRNA encoding a combination thereof.

[0212] In another embodiment, the present invention provides a method for inhibiting primary HSV infection in a subject, the method comprising administering to the subject the composition of the present invention. In another embodiment, the present invention provides a method for treating HSV infection in a subject, the method comprising administering to the subject the composition of the present invention. In another embodiment, the present invention provides a method for reducing the occurrence of HSV infection in a subject, the method comprising administering to the subject the composition of the present invention. In another embodiment, the present invention provides a method for inhibiting recurrence after primary HSV infection in a subject, the method comprising administering to the subject the composition of the present invention.

[0213] In one embodiment, the present invention provides a method for treating and / or suppressing primary HSV infection and / or secondary HSV infection. In one embodiment, "primary" infection refers to the first infection. In one embodiment, "secondary" infection refers to recurrence of HSV infection.

[0214] In one embodiment, "recrudescence" or "recurrence" refers to reinfection of skin tissue after HSV infection of latent nerve cells. In another embodiment, the term refers to reactivation of HSV after the latency period. In another embodiment, the term refers to symptomatic HSV lesions after an asymptomatic latency period.

[0215] In another embodiment, the present invention provides a method of inhibiting the spread of HSV. In one embodiment, the spread from DRG to skin is inhibited. In one embodiment, the cell-to-cell spread of HSV is inhibited. In one embodiment, anterograde spread is inhibited. In one embodiment, retrograde spread is inhibited. "DRG" refers to nerve cell bodies in one embodiment and contains nerve cell bodies of nerve fibers in another embodiment. In another embodiment, the term refers to any other definition of "DRG" used in the art. In another embodiment, the spread of HSV to nerve tissue is inhibited.

[0216] In another embodiment, the present invention provides a method of inhibiting recurrence after primary HSV infection in a subject, the method comprising the step of administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of preventing recurrence after primary HSV infection in a subject, the method comprising the step of administering to the subject a composition of the present invention.

[0217] In another embodiment, the present invention provides a method of inhibiting HSV labialis after primary HSV infection in a subject, the method comprising the step of administering to the subject a composition of the present invention.

[0218] In another embodiment, the present invention provides a method for preventing recurrence of HSV infection, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for treating the severity of recurrence of HSV infection, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for reducing the frequency of recurrence of HSV infection, the method comprising administering to the subject a composition of the present invention. In one embodiment, the present invention provides any of the methods described in an HIV-infected subject.

[0219] In another embodiment, the present invention provides a method for treating HSV encephalitis in a subject, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for reducing the occurrence of HSV encephalitis in a subject, the method comprising administering to the subject a composition of the present invention. "HSV encephalitis" refers to encephalitis caused by herpes simplex virus-1 (HSV) in one embodiment. In another embodiment, the term refers to encephalitis associated with HSV. In another embodiment, the term refers to any other type of HSV-mediated encephalitis known in the art.

[0220] In another embodiment, the present invention provides a method for treating or reducing HSV neonatal infection in a subject, the method comprising administering to the subject a composition of the present invention.

[0221] In another embodiment, the present invention provides a method for introducing an HSV glycoprotein into a cell of a subject, the method comprising contacting the cell with an in vitro transcribed mRNA molecule encoding a recombinant protein, the in vitro transcribed mRNA molecule further comprising a modified nucleoside, thereby introducing the HSV glycoprotein into the cell of the subject.

[0222] In another embodiment, the present invention provides a method of introducing mammalian cells to produce an HSV glycoprotein, the method comprising contacting the mammalian cells with an in vitro synthesized mRNA molecule encoding the HSV glycoprotein, wherein the in vitro synthesized mRNA molecule contains pseudouridine, thereby inducing the mammalian cells to produce the HSV glycoprotein.

[0223] References to HSV herein refer to HSV-1 in one embodiment, HSV-2 in another embodiment, and HSV-1 and HSV-2 in another embodiment.

[0224] "HSV-1" refers to herpes simplex virus-1 in another embodiment. 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.

[0225] "HSV-2" refers to herpes simplex virus-2 in another embodiment. In another embodiment, the term refers to the HSV-2 333 strain. In another embodiment, the term refers to the 2.12 strain. In another embodiment, the term refers to the HG52 strain. In another embodiment, the term refers to the MS strain. In another embodiment, the term refers to the G strain. In another embodiment, the term refers to the 186 strain. In another embodiment, the term refers to any other HSV-2 strain known in the art.

[0226] In another embodiment, the present invention provides a method of vaccinating a subject against HSV infection, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of suppressing HSV infection in a subject, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of interfering with HSV infection in a subject, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of interfering with primary HSV infection in a subject, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of interfering with HSV transmission in neurons of a subject, the method comprising administering to the subject a composition of the present invention.

[0227] The terms "interfering with HSV infection" and "interfering with primary HSV infection" refer, in another embodiment, to reducing the titer of infectious virus. In another embodiment, the terms refer to reducing the degree of viral replication.

[0228] In another embodiment, the present invention provides a method of reducing the occurrence of HSV-mediated herpes eye disease in a subject, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of treating HSV-1 corneal infection or herpes keratitis in a subject, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of reducing the occurrence of HSV-1 corneal infection or herpes keratitis in a subject, the method comprising administering to the subject a composition of the present invention.

[0229] In another embodiment, the present invention provides a method of treating, suppressing, or inhibiting HSV genital infection, comprising the step of administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of treating, suppressing, or inhibiting any symptom of recurrent HSV infection, comprising the step of administering to the subject a composition of the present invention.

[0230] In another embodiment, the present invention provides a method of reducing the occurrence of HSV-mediated genital ulcerative disease in a subject, comprising the step of administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of preventing the establishment of latent HSV infection in a subject, comprising the step of administering to the subject a composition of the present invention.

[0231] In one embodiment, the present invention provides a method of treating, suppressing, or inhibiting genital herpes infection in a subject, comprising the step of administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of treating, suppressing, or inhibiting oral herpes infection in a subject, comprising the step of administering to the subject a composition of the present invention.

[0232] In another embodiment, the present invention provides a method of reducing the occurrence of HSV-mediated encephalitis in a subject, comprising the step of administering to the subject a composition of the present invention.

[0233] In another embodiment, the herpes encephalitis treated or prevented by the method of the present invention is localized herpes encephalitis. In another embodiment, the herpes encephalitis is neonatal herpes encephalitis. In another embodiment, the herpes encephalitis is any other type of herpes encephalitis known in the art.

[0234] In another embodiment, the present invention provides a method for treating or reducing the occurrence of a disease, disorder, or symptom associated with or secondary to HSV encephalitis in a subject, the method comprising administering to the subject a composition of the present invention.

[0235] In another embodiment, the present invention provides a method for treating HSV infection in a subject, reducing the pathogenicity of HSV infection, restoring the symptoms of HSV infection, restoring the secondary symptoms of HSV infection, reducing the occurrence of HSV infection, or prolonging the latency until recurrence of HSV infection, the method comprising administering to the subject a composition of the present invention.

[0236] In another embodiment, the present invention provides a method for protecting a subject against the formation of herpes zoster lesions or similar epidemics in a human subject. In another embodiment, the present invention provides a method for inhibiting the formation of HSV herpes zoster lesions or similar epidemics in a human subject.

[0237] "Herpes zoster" refers, in one embodiment, in particular, to the skin lesion characteristics of HSV infection during reactivated infection, and in one embodiment, begins as a rash, subsequently distributes near the dermatome, and generally occurs in a strip or belt-like pattern. In one embodiment, the rash develops into small vesicles filled with vesicles or serous fluid. In one embodiment, herpes zoster lesions form in mice as a result of contact with HSV. In another embodiment, herpes zoster lesions form in humans as a result of contact with HSV. "Herpes zoster transmission" refers, in one embodiment, to HSV infection that spreads from a nodule into secondary skin sites within the dermatome. In another embodiment, the term refers to spread within the same dermatome as the site of initiation of the infection. In another embodiment, the term refers to any other definition of "herpes zoster transmission" known in the art. "Epidemic" refers, in another embodiment, to a sudden increase in the symptoms of a disease, or in the transmission or spread of a disease, and in one embodiment, to a sudden increase in herpes zoster lesions, while in another embodiment, "epidemic" refers to a sudden outburst of herpes zoster lesions.

[0238] In one embodiment, the present invention provides a method of interfering with the formation of dermatomal lesions or similar conditions in a subject. In one embodiment, the dermatomal lesions are formed as a result of contact with HSV. In another embodiment, the dermatomal lesions occur most frequently when the virus reactivates from latency in a nodule, and in one embodiment, the spread descends along a nerve and, in one embodiment, causes a reinfection.

[0239] It should be understood that the methods of the present invention can be used to treat, inhibit, suppress, etc. HSV infection or primary or secondary symptoms associated with such infection after exposure of a subject to HSV. In another embodiment, the subject is infected with HSV prior to vaccination. In another embodiment, the subject is at risk of HSV infection. In another embodiment, vaccination by the methods of the present invention is effective to treat, inhibit, suppress, etc. HSV infection or primary or secondary symptoms associated with such infection, regardless of whether the subject is infected with HSV at the time of vaccination.

[0240] In one embodiment, "treating" refers to a therapeutic or prophylactic or preventive treatment, the purpose of which is to prevent or alleviate a pathological condition or disorder targeted as described above herein. Thus, in one embodiment, treating may directly affect or cure, suppress, inhibit, prevent, reduce the severity of, delay the onset of, or reduce a disease, disorder or condition, or a combination thereof, and the associated symptoms. Thus, in one embodiment, "treating" particularly refers to delaying progression, facilitating remission, inducing remission, increasing remission, accelerating recovery, increasing the effectiveness of alternative treatments, or reducing resistance to alternative treatments, or a combination thereof. In one embodiment, "preventing" particularly refers to delaying the onset of symptoms, preventing recurrence of a disease, reducing the number or frequency of recurrence episodes, increasing the latency between symptomatic episodes, or a combination thereof. In one embodiment, "suppressing" or "inhibiting" particularly refers to reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the occurrence of symptoms associated with a disease, reducing the latency of symptoms, restoring symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[0241] In one embodiment, the compositions and methods of the invention are effective to reduce the rate of HSV acquisition, the duration of HSV infection, the frequency of HSV reactivation, or a combination thereof. In another embodiment, the compositions and methods of the invention are effective to treat or inhibit genital ulcerative disease and, in one embodiment, are required to reduce the severity or frequency of HSV genital ulcerative disease. In one embodiment, the compositions and methods of the invention block immune evasion from complement. In one embodiment, vaccination with HSV subunits encoded by mRNA can generate high titers of neutralizing antibodies or a strong T cell response, but during subsequent infection, HSV immune evasion molecules may block the activity of antibodies or T cells, thereby reducing the effectiveness of the composition. In one embodiment, the compositions and methods of the invention incorporate a strategy to block virus-mediated immune evasion, for example, by using gC-1 to prevent immune evasion from complement and enhance the effectiveness of, for example, a gD-1 subunit composition.

[0242] In one embodiment, studies in guinea pigs and mice suggest that the amount of virus in nodules correlates with the frequency of recurrent HSV infection. Thus, in one embodiment, the compositions and methods of the invention are useful to prevent or inhibit recurrent HSV infection. In one embodiment, for example, antibodies to gC-1 block domains involved in immune evasion, enhance complement activity, improve the neutralizing activity against gD-1, increase antibody- and complement-dependent cytotoxic activity, and increase complement-mediated neutralization and lysis of infected cells.

[0243] In one embodiment, the symptom is primary, while in another embodiment, the symptom is secondary. In one embodiment, "primary" refers to a symptom that is a direct result of a viral infection in a subject, while in one embodiment, "secondary" refers to a symptom that is caused by or is a result of the primary cause. In one embodiment, the compositions and strains for use in the present invention treat primary or secondary symptoms or secondary complications associated with HSV infection.

[0244] In another embodiment, a "symptom" may be any sign of HSV infection, including blisters, ulcers, or lesions on the urethra, cervix, upper thigh, and / or anus in women or on the penis, urethra, scrotum, upper thigh, and anus in men, inflammation, swelling, fever, influenza-like symptoms, stomatitis, pharyngitis, tonsillitis, pain, edema, ulcers, cold sores, neck pain, swollen lymph nodes, redness, bleeding, itching, dysuria, headache, myalgia, or combinations thereof.

[0245] In another embodiment, the disease, disorder, or symptom is fever. In another embodiment, the disease, disorder, or symptom is headache. In another embodiment, the disease, disorder, or symptom is shoulder pain. In another embodiment, the disease, disorder, or symptom is seizure. In another embodiment, the disease, disorder, or symptom is hemiplegia. In another embodiment, the disease, disorder, or symptom is coma. In another embodiment, the disease, disorder, or symptom is stupor. In another embodiment, the disease, disorder, or symptom is any other disease, disorder, or symptom known in the art that is associated with or secondary to herpes encephalitis.

[0246] Methods for determining the presence and severity of herpes encephalitis are well known in the art, for example, Bonkowsky JL et al., (Herpes simplex virus central nervous system relapse during treatment of infantile spasms with corticotropin, Pediatrics, May 2006; 117(5): e1045-1048) and Khan OA et al., (Herpes encephalitis presenting as mild aphasia: case report, BMC FamPract, March 24, 2006; 7: 22). Each method represents a separate embodiment of the present invention.

[0247] In another embodiment, the present invention provides a method for treating or reducing the occurrence of a disease, disorder, or symptom associated with HSV infection in a subject, the method comprising administering to the subject a composition of the present invention.

[0248] In another embodiment, the disease, disorder, or symptom secondary to HSV infection is oral lesions. In another embodiment, the disease, disorder, or symptom is genital lesions. In another embodiment, the disease, disorder, or symptom is oral ulcers. In another embodiment, the disease, disorder, or symptom is genital ulcers. In another embodiment, the disease, disorder, or symptom is fever. In another embodiment, the disease, disorder, or symptom is headache. In another embodiment, the disease, disorder, or symptom is muscle pain. In another embodiment, the disease, disorder, or symptom is enlarged glands in the groin. In another embodiment, the disease, disorder, or symptom is painful urinary passage. In another embodiment, the disease, disorder, or symptom is vaginal discharge. In another embodiment, the disease, disorder, or symptom is blistering. In another embodiment, the disease, disorder, or symptom is flu-like malaise. In another embodiment, the disease, disorder, or symptom is keratitis. In another embodiment, the disease, disorder, or symptom is herpetic whitlow. In another embodiment, the disease, disorder, or symptom is Bell's palsy. In another embodiment, the disease, disorder, or symptom is herpetic erythema multiforme. In another embodiment, the disease, disorder, or symptom is a lower back symptom (e.g., numbness, tingling in the buttocks or perianal area, urinary retention, constipation, and impotence). In another embodiment, the disease, disorder, or symptom is localized herpetic eczema. In another embodiment, the disease, disorder, or symptom is disseminated herpetic eczema. In another embodiment, the disease, disorder, or symptom is combat herpes. In another embodiment, the disease, disorder, or symptom is herpetic balanitis. In another embodiment, the disease, disorder, or symptom is an esophageal symptom (e.g., difficult or burning swallowing, tight throat pain while swallowing, weight loss, pain in the upper chest or back while swallowing). In another embodiment, the disease, disorder, or symptom is any other disease, disorder, or symptom known in the art. Each disease, disorder, and condition represents a separate embodiment of the present invention.

[0249] Accordingly, in one embodiment, the compositions and methods of the present invention treat, suppress, inhibit, or reduce the occurrence of the infection itself, while in another embodiment, the compositions and methods of the present invention treat, suppress, inhibit, or reduce the occurrence of the initial symptoms of the infection, while in another embodiment, the compositions and methods of the present invention treat, suppress, inhibit, or reduce the occurrence of the secondary symptoms of the infection. It should be understood that the compositions and methods of the present invention can affect any combination of the infection, the initial symptoms caused by the infection, and the secondary symptoms associated with the infection.

[0250] The HSV infections treated or improved by the methods and compositions of the present invention are, in another embodiment, genital HSV infections. In another embodiment, the HSV infection is an oral HSV infection. In another embodiment, the HSV infection is an ocular HSV infection. In another embodiment, the HSV infection is a dermatological HSV infection.

[0251] In another embodiment, the present invention provides a method for reducing the occurrence of disseminated HSV infection in a subject, the method comprising administering to the subject a composition of the present invention.

[0252] In another embodiment, the present invention provides a method for reducing the occurrence of neonatal HSV infection in the offspring of a subject, the method comprising administering to the subject a composition of the present invention.

[0253] In another embodiment, the present invention provides a method for reducing the transmission of HSV infection from a subject to its offspring, the method comprising administering to the subject a composition of the present invention.

[0254] In another embodiment, the offspring is an infant. In another embodiment, the transmission that is reduced or inhibited is perinatal transmission. In another embodiment, the transmission during breastfeeding is reduced or inhibited. In another embodiment, the transmission that is reduced or inhibited is any other type of parent-to-offspring transmission known in the art.

[0255] In another embodiment, the present invention provides a method for reducing the severity of neonatal HSV infection in an offspring of a subject, the method comprising administering to the subject a composition of the present invention.

[0256] In one embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV infection in an HIV-infected subject, the method comprising administering to the subject a composition comprising (a) a modified mRNA encoding an HSV gC protein or a fragment thereof; (b) a modified mRNA encoding an HSV gE protein or a fragment thereof; and (c) an adjuvant. In another embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the occurrence of HSV infection in an HIV-infected subject, the method comprising administering to the subject a composition comprising (a) a modified mRNA encoding an HSV gC protein or a fragment thereof, the fragment comprising any of its C3b-binding domain, its properdin interference domain, its C5 interference domain, or a fragment of its C3b-binding domain, properdin interference domain, or C5 interference domain; (b) a modified mRNA encoding an HSV gE protein or a fragment thereof, the fragment comprising AA 24-409 or a fragment thereof; and (c) an adjuvant.

[0257] In another embodiment, the present invention provides a method for treating HSV infection in a subject infected with HIV, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for suppressing HSV infection in a subject infected with HIV, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for inhibiting HSV infection in a subject infected with HIV, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for reducing the occurrence of HSV infection in a subject infected with HIV, the method comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for preventing HIV infection, the method comprising administering to the subject an HSV composition of the present invention. In one embodiment, HSV infection increases the risk of HIV infection and protection against HSV infection decreases the risk of HIV infection. Thus, in one embodiment, the present invention provides a method for reducing the risk of HIV infection, the method comprising administering to the subject a composition of the present invention.

[0258] In one embodiment, the composition for use in the method of the present invention elicits an immune response against HSV. In another embodiment, the composition for use in the method of the present invention elicits an immune response against HSV-1. In another embodiment, the composition for use in the method of the present invention elicits an immune response against HSV-2. In another embodiment, the composition comprises modified mRNA encoding gD and gC proteins. In another embodiment, the composition comprises modified mRNA encoding gE and gD proteins. In another embodiment, the composition comprises modified mRNA encoding gC and gE proteins. In another embodiment, the composition comprises modified mRNA encoding gE, gD, and gC proteins. In another embodiment, the composition comprises modified mRNA encoding gE, gD, or gC proteins. In another embodiment, the protein encoded by the modified mRNA is an HSV-1 protein. In another embodiment, the protein encoded by the modified mRNA is an HSV-2 protein. In another embodiment, the protein encoded by the modified mRNA comprises both HSV-1 and HSV-2 proteins.

[0259] In one embodiment, it should be understood that a subject according to any of the embodiments described herein may be a subject infected with HSV, or in another embodiment, a subject suspected of being infected with HSV. In one embodiment, the subject may be infected with at least one other pathogen, or in another embodiment, suspected of being infected with at least one other pathogen. In one embodiment, the subject may be susceptible. In one embodiment, the subject is infected with HSV, while in another embodiment, the subject is at risk of infection by HSV, in one embodiment, a subject who is a neonate, in another embodiment, susceptible, in another embodiment, an elderly person, and in another embodiment, a susceptible neonate or susceptible elderly subject.

[0260] In another embodiment, the compositions of the invention and their related uses can suppress, inhibit, prevent, or treat HIV infection in a subject. In one embodiment, the compositions of the invention and their related uses can treat secondary complications of HIV infection, which in one embodiment are opportunistic infections, neoplasms, neurological abnormalities, or progressive immune decline. In another embodiment, the method includes the step of treating acquired immunodeficiency syndrome (AIDS). In another embodiment, the method includes the step of treating a decrease in the number of CD4 + T lymphocytes.

[0261] In another embodiment, the invention provides a method for reducing HIV-1 transmission to offspring, the method comprising administering a composition of the invention to a subject. As is known in the art, HSV-2 infection increases the excretion of HIV-1 virus in genital secretions (Nagot N et al., Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus, N Engl J Med., February 22, 2007; 356(8):790-799). Thus, the method of the invention for inhibiting HSV-2 infection is also effective for reducing HIV-1 transmission to offspring. In another embodiment, the mutant HSV strain is an HSV-1 strain. In another embodiment, the mutant HSV strain is an HSV-2 strain.

[0262] In another embodiment, the invention provides a method for reducing HIV-1 transmission to a sexual partner, the method comprising administering a composition of the invention to a subject. As is known in the art, HSV-2 infection increases the excretion of HIV-1 virus in genital secretions. Thus, the method of the invention for inhibiting HSV-2 infection is also effective for reducing HIV-1 transmission to a sexual partner. In another embodiment, the mutant HSV strain is an HSV-1 strain. In another embodiment, the mutant HSV strain is an HSV-2 strain.

[0263] In another embodiment, the present invention provides a method for reducing susceptibility to HIV-1, the method comprising administering to a subject the composition of the present invention. As is known in the art, HSV-2 infection increases HIV-1 replication (Ouedraogo A et al., Impact of suppressive herpes therapy on genital HIV-1 RNA among women taking antiretroviral therapy: a randomized controlled trial, AIDS, November 28, 2006; 20(18):2305-13). Thus, the method of the present invention that inhibits HSV-2 infection is also effective in reducing susceptibility to HIV-1. In another embodiment, the mutant HSV strain is an HSV-1 strain. In another embodiment, the mutant HSV strain is an HSV-2 strain.

[0264] Accordingly, in one embodiment, the present invention provides a method for inhibiting primary HSV infection in an HIV-infected subject, the method comprising administering to the subject the composition of the present invention. In another embodiment, the present invention provides a method for reducing the occurrence of HSV infection in an HIV-infected subject, the method comprising administering to the subject the composition of the present invention. In another embodiment, the present invention provides a method for inhibiting relapse, recurrence, or HSV labialis after primary HSV infection in an HIV-infected subject, the method comprising administering to the subject the composition of the present invention. In one embodiment, administration of the composition of the present invention induces an anti-HSV immune response.

[0265] In another embodiment, the present invention provides a method of inducing an immune response in a subject, the method comprising administering to the subject an mRNA composition in which the nucleosides of the present invention are modified. In another embodiment, the immune response includes a CD4 immune response. In another embodiment, the immune response includes a CD8 immune response. In another embodiment, the immune response includes a follicular helper T cell immune response. In another embodiment, the immune response includes a germinal center B cell immune response. In another embodiment, the immune response includes an IgG antibody response against gC2, gD2, gE2, or a combination thereof.

[0266] In another embodiment, the present invention provides a method of treating herpes simplex virus (HSV) infection in a subject, the method comprising intramuscularly administering to the subject an mRNA composition in which the nucleosides of the present invention are modified. In another embodiment, the present invention provides a method of suppressing, inhibiting, or reducing the occurrence of herpes simplex virus (HSV) infection in a subject, the method comprising intramuscularly administering to the subject an mRNA composition in which the nucleosides of the present invention are modified.

[0267] Administration and Pharmaceutical Regimen In another embodiment, the composition of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, vaginally, nasally, intratumorally, or topically.

[0268] In another embodiment, "administering" refers to directly introducing the composition of the present invention into a subject by injection or other means. In another embodiment, "administering" refers to contacting cells of the subject's immune system with the composition or a modified mRNA encoding an HSV protein or a mixture thereof.

[0269] In another embodiment of the method and composition of the present invention, the composition is administered orally and is thus formulated in a form suitable for oral administration, i.e., a solid or liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets, and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, and the like. In another embodiment of the present invention, the active ingredient is formulated within a capsule. In accordance with this embodiment, the composition of the present invention includes a hard gelatin capsule in addition to the active compound, inert carrier or diluent.

[0270] In other embodiments, the pharmaceutical composition is administered by intravenous, intra - arterial, or intramuscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils, and the like. In another embodiment, the pharmaceutical composition is administered intravenously and is thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intra - arterially and is thus formulated in a form suitable for intra - arterial administration. In another embodiment, the pharmaceutical composition is administered intramuscularly and is thus formulated in a form suitable for intramuscular administration.

[0271] In another embodiment, the pharmaceutical composition is administered topically to the body surface and is thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, eye drops, and the like. For topical administration, the composition or their physiologically acceptable derivatives are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent, with or without a pharmaceutical carrier.

[0272] In another embodiment, the composition is administered as a suppository, e.g., a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of pellets. In another embodiment, the pellets provide for a controlled release of the drug over a long period of time.

[0273] In a preferred embodiment, the pharmaceutical composition is administered intramuscularly, subcutaneously or intradermally.

[0274] The "effective dosage" of the modified mRNA refers, in another embodiment, to an amount sufficient to exert a therapeutic effect. In another embodiment, the term refers to an amount sufficient to induce the expression of a detectable amount of the encoded protein. Each possibility represents a separate embodiment of the invention.

[0275] Methods for measuring the dosage of modified mRNA encoding an HSV glycoprotein (e.g., in a human subject) are well known in the art and include, for example, dose escalation studies. Each method represents a separate embodiment of the invention.

[0276] In some embodiments, either the HSV composition of the invention or the HSV composition for use in the method of the invention comprises a modified mRNA encoding an HSV protein of the invention or a combination of modified mRNAs encoding HSV proteins in any form or embodiment described herein. In some embodiments, either the composition and the composition for use in the method of the invention comprises a modified mRNA encoding an HSV protein of the invention or a combination of modified mRNAs encoding HSV proteins in any form or embodiment described herein. In some embodiments, the composition of the invention consists essentially of, in any form or embodiment described herein, a modified mRNA encoding an HSV protein of the invention or a combination of modified mRNAs encoding HSV proteins. In some embodiments, the term "comprising" refers to the inclusion of modified mRNAs encoding other HSV proteins as well as modified mRNAs encoding other proteins that may be known in the art. In some embodiments, the term "consisting essentially of" refers to a composition having a modified mRNA encoding a particular HSV protein or fragment thereof. However, other components that do not directly contribute to the utility of the modified mRNA(s) encoding the HSV protein(s) may be included. In some embodiments, the term "consisting of" refers to a composition having, in any form or embodiment described herein, a modified mRNA encoding a particular HSV protein or fragment, or a combination of modified mRNAs encoding HSV proteins or fragments of the invention.

[0277] In another embodiment, the invention provides a composition for treating HSV-1 or its symptoms or signs comprising the modified mRNA of the invention.

[0278] In another embodiment, the present invention provides a composition for treating HSV-2 or its symptoms or signs, comprising a modified mRNA of the present invention.

[0279] In one embodiment, the compositions and methods of the present invention can be used similarly in non-HSV herpesviruses that are, in one embodiment, the gD, gE, or gC protein of HSV-1, or in another embodiment, the gD, gE, or gC protein of HSV-2 that is, in one embodiment, the gD, gE, or gC protein of HSV-1, or in another embodiment, the gD, gE, or gC protein of HSV-2, and that are 70% homologous, in another embodiment 80% homologous, in another embodiment 85% homologous, in another embodiment 90% homologous, in another embodiment 95% homologous, in another embodiment 98% homologous, and in another embodiment 100% homologous. In one embodiment, such compositions can be useful for suppressing, inhibiting, preventing, or treating cancer, or in another embodiment, tumors. In one embodiment, non-HSV herpesviruses include varicella-zoster virus (VZV), Epstein-Barr virus (EBV), EBNA, cytomegalovirus (CMV), and human herpesvirus-6 (HHV-6).

[0280] In another embodiment of the method of the present invention, the composition of the present invention is administered once. In another embodiment, the composition is administered twice. In another embodiment, the composition is administered three times. In another embodiment, the composition is administered four times. In another embodiment, the composition is administered at least four times. In another embodiment, the composition is administered more than four times.

[0281] In another embodiment, the dosage is a daily dosage. In another embodiment, the dosage is a once-weekly dosage. In another embodiment, the dosage is a once-monthly dosage. In another embodiment, the dosage is a once-yearly dosage. In another embodiment, the dosage is a series of a defined number of dosages. In another embodiment, the dosage is a single dosage.

[0282] In one embodiment, any of the booster administrations described above herein are administered after a priming dose comprising one or more modified mRNAs encoding an HSV-1 protein or an immunogenic fragment thereof. In another embodiment, any of the booster administrations described above herein are administered after a priming vaccination comprising one or more modified mRNAs encoding an HSV-2 protein or an immunogenic fragment thereof.

[0283] In one embodiment, the subject is immunized with a single administration of the composition. In another embodiment, the subject is immunized with a single administration. In another embodiment, the subject is immunized with two administrations. In another embodiment, the subject is immunized with three administrations. In another embodiment, the subject is immunized with four administrations. In another embodiment, the subject is immunized with five administrations.

[0284] In one embodiment, all components of the composition are provided at equal concentrations. In this aspect, in one embodiment, the modified mRNAs encoding gC, gD, and gE are provided in a 1:1:1 ratio. In another embodiment, the modified mRNAs encoding gC, gD, and gE are provided in a 5:2:5 ratio. In another embodiment, the modified mRNAs encoding gC and gD are provided in a 1:1 ratio. In another embodiment, the modified mRNAs encoding gC and gE are provided in a 1:1 ratio. In another embodiment, the modified mRNAs encoding gD and gE are provided in a 1:1 ratio.

[0285] In one embodiment, the modified mRNA, combined with gC, gD, gE, or a combination thereof, or with other HSV glycoproteins, is administered in the same composition, at the same site, and by the same route, while in another embodiment, the modified mRNAs encoding gC, gD, and gE are administered in separate compositions, at separate sites but by the same administration route, or in another embodiment, the modified mRNAs encoding gC, gD, and gE are administered in separate compositions, at separate sites, by different administration routes, or in another embodiment, the modified mRNAs encoding gC, gD, and gE are administered in separate compositions, at the same site, by different administration routes (e.g., injection and topical).

[0286] In one embodiment, the method of the invention comprises a single or one-time administration of a composition comprising one or more nucleoside-modified mRNAs of the invention. In another embodiment, the method of the invention comprises administration of a composition comprising one or more nucleoside-modified mRNAs in a prime and boost approach. In one embodiment, the method of the invention further comprises administering to the subject one or more additional administrations of the nucleoside-modified mRNA composition following the first administration.

[0287] In another embodiment, the method of the invention comprises administering, as a first administration, a composition comprising one or more nucleoside-modified mRNAs encoding one or more HSV glycoproteins and, as a second or consecutive administration, a composition comprising one or more HSV glycoproteins. In one embodiment, the HSV glycoprotein encoded by the mRNA in the first (or initial) administration is the same glycoprotein in the second or consecutive (or boost) administration. In another embodiment, a composition comprising one or more HSV glycoproteins is administered as the first administration and a composition comprising one or more nucleoside-modified mRNAs encoding one or more HSV glycoproteins is administered as the second or consecutive administration. Each possibility represents a separate embodiment of the invention.

[0288] In another embodiment, the modified mRNAs encoding gC, gD, and gE are co-administered following a booster administration of the modified mRNA encoding gD that does not include the modified mRNA encoding gC or gE. In another embodiment, the modified mRNAs encoding gC, gD, and gE are co-administered following a booster administration of the modified mRNA encoding gC that does not include the modified mRNA encoding gD or gE. In another embodiment, the modified mRNAs encoding gC, gD, and gE are co-administered following a booster administration of the modified mRNA encoding gE that does not include the modified mRNA encoding gD or gC. In another embodiment, the modified mRNAs encoding gC, gD, and gE are co-administered following a booster administration of the modified mRNAs encoding gC and gD that do not include the modified mRNA encoding gE. In another embodiment, the modified mRNAs encoding gC, gD, and gE are co-administered following a booster administration of the modified mRNAs encoding gC and gE that do not include the modified mRNA encoding gD. In another embodiment, the modified mRNAs encoding gC, gD, and gE are co-administered following a booster administration of the modified mRNAs encoding gD and gE that do not include the modified mRNA encoding gE. In one embodiment, the booster administration is performed at the same site and by the same mode of administration as the priming dose. In another embodiment, the booster administration is performed at a different site than the priming dose but by the same mode of administration as the priming dose. In one embodiment, the booster administration is performed at the same site as the priming dose but by a different mode of administration. In another embodiment, the booster administration is performed at a different site and by a different mode of administration than the priming dose.

[0289] In one embodiment, the modified mRNA induces a detectably lower innate immune response than unmodified RNA having the same amount of the same sequence.

[0290] In one embodiment, the effectiveness of the compositions and methods of the present invention depends on the presence of complement, while in another embodiment, the compositions and methods of the present invention are independent of the presence of complement. In one embodiment, the effectiveness of some of the compositions for use in the methods of the present invention depends on the presence of complement, while others do not. In one embodiment, the anti-gC antibody is complement-dependent for its effectiveness against HSV.

[0291] In one embodiment, complement is an important participating factor for both innate and acquired immunity. In one embodiment, complement activation promotes virus neutralization by phagocytosis and lysis, functions as a chemoattractant for neutrophils and macrophages, and enhances B and T cell responses. In one embodiment, HSV-1 gC binds to complement C3b, blocks the properdin interaction with C5 and C3b, and inhibits complement activation and complement-mediated virus neutralization. In one embodiment, the gC-1 domain that interacts with complement is located within amino acids 33-133, blocks properdin binding to C5 and C3b, and in one embodiment, the gC-1 domain that interacts with complement extends from amino acids 124-366 and binds directly to C3b. In one embodiment, an HSV-1 gC mutant virus lacking the C3b binding domain is sensitive to complement-mediated virus neutralization in vitro and is less pathogenic than the wild-type (WT) virus in the mouse flank model. Therefore, in one embodiment, the interaction between gC-1 and C3b enhances HSV-1 pathogenicity, and in one embodiment, blocking of the gC-1 domain is effective in the prevention or treatment of HSV-1 infection.

[0292] In one embodiment, the compositions and methods of the invention are for use in a human subject, while in another embodiment, they are for use in an animal subject. In another embodiment, the subject is a mammal. In another embodiment, the subject is any organism suspected of being infected by HSV. In one embodiment, the subject is a mouse, cow, sheep, dog, cat, horse, pig, etc. In one embodiment, the compositions and methods of the invention are effective in a male subject. In another embodiment, the compositions and methods of the invention are effective in a female subject. In one embodiment, the compositions and methods of the invention are effective in a seronegative subject. In another embodiment, the compositions and methods of the invention are effective in a seropositive subject.

[0293] Pharmaceutical preparation In one embodiment, the method of the invention further comprises the step of mixing the modified mRNA with a transfection reagent prior to the contacting step. In another embodiment, the method of the invention further comprises the step of administering the modified mRNA together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0294] 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.

[0295] In another embodiment, the transfection reagent forms liposomes. The liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency, and improve biological activity.

[0296] In another embodiment, the liposomes are hollow spherical vesicles containing lipids arranged in the same manner as these lipids that make up the cell membrane. These have, in another embodiment, an internal aqueous space for encapsulating water-soluble compounds and are in the size range of 0.05 to several microns in diameter. In another embodiment, the liposomes can deliver RNA to cells in a biologically active form (see Langer, Science 249:1527-1533 (1990); Treat et al., in 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.).

[0297] Each type of transfection reagent represents a separate embodiment of the present invention.

[0298] In another embodiment, the modified mRNA of the present invention is encapsulated in nanoparticles. Nanoparticle packaging methods are well known in the art, for example, Bose S et al., (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells, J. Virol., 78:8146, 2004); Dong Y et al., Poly(d,l-lactide-co-glycolide) / montmorillonite nanoparticles for oral delivery of anticancer drugs, Biomaterials, 26:6068, 2005); Lobenberg R et al. (Improved body distribution of 14C-labelled AZT bound to nanoparticles in rats determined by radio luminography, J Drug Target, 5:171, 1998); Sakuma S R et al. (Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract, Int J Pharm, 177:161, 1999); Virovic L et al., (Novel delivery methods for treatment of viral hepatitis: an update, Expert Opin Drug Deliv, 2:707, 2005); and Zimmermann E et al., (Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media, Eur J Pharm Biopharm, 52:203, 2001). Each method represents a separate embodiment of the present invention.

[0299] In one embodiment, ψmRNA is encapsulated in nanoparticles to improve delivery efficiency and ψmRNA expression. Nanoparticle packaging involves concentrating the RNA and encapsulating it in particles smaller than the pores of the nuclear membrane using chemicals including poly-L-lysine and polyethylene glycol. In one embodiment, the RNA is packaged in one of four nanoparticle formulations (PEI, PLL, PAE, and CK 30 PEG 10k ).

[0300] Lipid nanoparticles In one embodiment, the nanoparticles used in the compositions and methods of the present invention include lipid nanoparticles as described in Cullis, P., and Hope, M, (undated), Lipid Nanoparticle Systems for Enabling Gene Therapies. Molecular therapy., 25(7), which is hereby incorporated by reference in its entirety.

[0301] In one embodiment, delivery of nucleoside-modified RNA includes any suitable delivery method, including typical RNA transfection methods described elsewhere herein. In certain embodiments, delivery of nucleoside-modified RNA to a subject includes mixing the nucleoside-modified RNA with a transfection reagent prior to the contacting step. In another embodiment, the method of the present invention further includes administering the nucleoside-modified RNA together with a transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0302] 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.

[0303] In another embodiment, the transfection reagent forms liposomes.

[0304] In another embodiment, the liposomes increase intracellular stability, increase uptake efficiency, and improve biological activity. In another embodiment, the liposomes are hollow spherical vesicles containing lipids arranged in the same manner as these lipids that make up the cell membrane. In another embodiment, they have an internal aqueous space for encapsulating water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, the liposomes can deliver RNA to cells in a biologically active form.

[0305] In one embodiment, the composition comprises lipid nanoparticles (LNPs) and one or more nucleic acid molecules described herein. For example, in one embodiment, the composition comprises LNPs and one or more modified RNA molecules encoding one or more antigens, an adjuvant, or a combination thereof.

[0306] The term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer order (e.g., 1 to 1,000 nm) and comprising one or more lipids, such as lipids of formula (I), (II) or (III) described in WO 2016 / 176330 A1, which is incorporated herein by reference in its entirety.

[0307] In some embodiments, the lipid nanoparticle is included in a formulation comprising RNA modified with the nucleosides described herein. In some embodiments, such lipid nanoparticles comprise one or more excipients selected from cationic lipids as well as neutral lipids, charged lipids, steroids and polymer-conjugated lipids (e.g., pegylated lipids of structure (IV) such as compound IVa). In some embodiments, the RNA modified with the nucleosides is encapsulated in an aqueous space covered by the lipid portion of the lipid nanoparticle or a part or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic cleavage or other undesired actions induced by the host organism or cell machinery, e.g., a harmful immune response.

[0308] 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 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 certain embodiments, the RNA modified with the nucleosides is resistant to degradation with nucleases when present in the lipid nanoparticles in an aqueous solution.

[0309] The LNP may comprise any lipid capable of forming particles to which one or more nucleic acid molecules bind or in which one or more nucleic acid molecules are encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters), are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids" including fats, oils, and waxes; (2) "compound lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0310] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0311] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term "cationic lipid" refers to a lipid that is cationic or becomes cationic (protonated) because the pH is lower than the pK of the ionic group of the lipid, but becomes increasingly more neutral at higher pH values. At pH values below the pK, the lipid can then bind to negatively charged nucleic acids. In certain embodiments, the cationic lipid includes zwitterionic lipids that are considered positively charged as the pH decreases.

[0312] In certain embodiments, the cationic lipid comprises any of a number of lipid moieties having a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N’,N’-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), octadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). In addition, a number of commercially available preparations of cationic lipids that can be used in the present invention are available. These include, for example, LIPOFECTIN® (cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), commercially available from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (cationic liposomes containing N-(l-(2,3-dioleoyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), commercially available from GIBCO / BRL); and TRANSFECTAM® (cationic lipids containing dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol, commercially available from Promega Corp., Madison, Wis.).The following lipids are cationic and have a positive charge at a pH below physiological pH.

[0313] DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0314] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the present invention include those described in International Publication No. WO 2012 / 016184, which is hereby incorporated by reference in its entirety. Representative amino lipids include 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-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), but are not limited thereto.

[0315] In certain embodiments, the cationic lipid is present in the LNP in an amount of about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP contains only the cationic lipid. In certain embodiments, the LNP contains one or more additional lipids that stabilize particle formation during their formation.

[0316] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0317] The term "neutral lipid" refers to any one of a number of lipid species that exist either uncharged or in a neutral zwitterionic form at physiological pH.

[0318] Representative neutral lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, and cerebroside.

[0319] Typical neutral lipids include, for example, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), dipalmitoyl oleoyl phosphatidylcholine (POPC), dipalmitoyl oleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC).

[0320] In some embodiments, the LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., the lipid of formula (I)) to the neutral lipid ranges from about 2:1 to about 8:1.

[0321] In various embodiments, the LNP further comprises a steroid or steroid analog.

[0322] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., the lipid of formula (I)) to cholesterol ranges from about 2:1 to 1:1.

[0323] The term "anionic lipid" refers to any lipid that is negatively charged 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 attached to neutral lipids.

[0324] In certain embodiments, the LNP comprises a glycolipid (e.g., monosialoganglioside Gmi). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

[0325] In some embodiments, the LNP comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule that includes 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 that includes both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art and include 1-(monomethylmethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-s-DMG), etc.

[0326] In certain embodiments, the LNP comprises an additional stabilizing lipid that is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

[0327] Representative polyethylene glycol lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol lipid is N-[(methoxypoly(ethylene glycol)2OOO)carbamyl]-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol lipid is PEG-c-DOMG. In other embodiments, the LNP comprises a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate such as Q-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 25:1.

[0328] In certain embodiments, the additional lipid is present in the LNP in an amount of about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 mole percent or about 1.5 mole percent.

[0329] In certain embodiments, the LNP comprises one or more targeting moieties capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor found on the cell surface.

[0330] In certain embodiments, the LNP comprises one or more internal translocation domains. For example, in one embodiment, the LNP comprises one or more domains that bind to a cell and induce internal translocation of the LNP. For example, in one embodiment, the one or more internal translocation domains bind to receptors found on the lipid surface and induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding to a molecule in vivo, whereupon the LNP-bound molecule can then be recognized by a cell surface receptor and induce internal availability. For example, in one embodiment, the LNP binds to systemic ApoE and induces uptake of the LNP and associated cargo.

[0331] Other exemplary LNPs and their manufacture are described in the art, for example, in International Publication No. WO 2016 / 176330 A1, US Patent Publication No. US 2012 / 0276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7):1357-1364; Basha et al., 2011, Mol Ther, 19(12):2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34):18440-18450; Lee et al., 2012, Int J Cancer., 131(5):E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1:e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34):8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids., 2, e139; Maier et al., 2013, Mol Ther., 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.

[0332] In another embodiment, the method of the present invention comprises administering a modified mRNA encoding an HSV glycoprotein and a pharmaceutically acceptable carrier or diluent. In other embodiments, the pharmaceutically acceptable carrier for a liquid formulation may be an aqueous or non-aqueous solution, suspension, emulsion or oil. Examples of non-aqueous solvents are injectable organic esters such as propylene glycol, polyethylene glycol, and ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish liver oil.

[0333] As used herein, "pharmaceutically acceptable carrier or diluent" is well known to those skilled in the art.

[0334] In another embodiment, the pharmaceutical composition provided herein is a controlled release composition, i.e., a composition in which the compound is released over a long period of time after administration. Release control or sustained release compositions include formulations in lipid-soluble depots (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate release composition, i.e., a composition in which the entire compound is released immediately upon administration.

[0335] Each of the additives, excipients, formulations and methods of administration represents a separate embodiment of the present invention.

[0336] In another embodiment, the present invention provides a kit comprising reagents utilized in the practice of the method of the present invention. In another embodiment, the present invention provides a kit comprising the composition, tool, or instructions of the present invention.

[0337] The following examples are presented to more fully illustrate preferred embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention.

Examples

[0338] Example 1: Materials and Experimental Methods Modified mRNAs expressing the extracellular domains of HSV-2 glycoproteins C, D, and E (gC2 / gD2 / gE2). Modified mRNAs (encoding gC2 (SEQ ID NO: 10), encoding gD2 (SEQ ID NO: 4), and encoding gE2 (SEQ ID NO: 16)) were prepared based on the DNA coding sequences encoding HSV-2 glycoprotein C (gC2) amino acids 27-426 (SEQ ID NO: 11) from HSV-2 strain 333, glycoprotein D (gD2) amino acids 26-331 (SEQ ID NO: 5) from HSV-2 strain 333, and glycoprotein E (gE2) amino acids 24-405 (SEQ ID NO: 17) from HSV-2 strain 2.12.

[0339] The modified mRNAs were incorporated into liposome nanoparticles (LNP) by Acuitas Therapeutics, and the following immunogens were prepared: (a) multivalent C mRNA in LNP; (b) gC2-modified mRNA in LNP; (c) gD2-modified mRNA in LNP; (d) gE2-modified mRNA in LNP; (e) gC2, gD2, and gE2-modified mRNA in LNP.

[0340] The immunization groups were as follows: a) Control (multivalent C group): Multivalent C mRNA / LNP divided into 4 aliquots and administered at 4 separate sites. b) gD2 alone (gD2 group): 10 μg of gD2 mRNA / LNP divided into 4 aliquots and administered at 4 separate sites. c) Individual trivalent (trivalent-I group): 10 μg of gC2 mRNA / LNP, 10 μg of gD2 mRNA / LNP, and 10 μg of gE2 mRNA / LNP, each divided into 2 aliquots and administered at 2 sites respectively. d) Combined trivalent (trivalent-C group): 10 μg of gC2 mRNA, 10 μg of gD2 mRNA, and 10 μg of gE2 mRNA combined in LNP, divided into 4 aliquots and administered at 4 separate sites.

[0341] Experimental method. Using an electric laser and a razor, the hair on the backs of 6 - 8 - week - old BALB / c mice was removed. Prior to the first and second immunizations, and prior to vaginal challenge, the mice were bled. The two immunizations were performed intradermally at 28 - day intervals. The intradermal immunization was performed on the exposed back. Five mice (upper group c) that received the trivalent vaccine at each site were sacrificed 14 days after the second immunization. Spleens were harvested for CD4 + and CD8 + T - cell responses. Twenty - eight days after the second immunization, the mice were treated subcutaneously with 2 mg of Depo - Provera, and 5 days later, 5×10 3 PFU of HSV - 2 strain MS (about 400 LD 50 ) were used to infect the mice vaginally. On days 2 and 4 after challenge, vaginal swabs were obtained for virus culture. On day 4 after challenge, several mice in each vaccination group were sacrificed, and the dorsal root ganglia (DRG) were excised for HSV - 2 DNA qPCR. The remaining animals were evaluated for 10 - day weight loss and hind - limb weakness, while survival and genital disease were monitored for 28 days.

[0342] Example 2: Characteristics of the translation products produced by gC2, gP2, and gE2 - modified mRNAs When transfected into mammalian cells, the ability of the modified mRNAs to express proteins of the expected molecular weight was verified. 0.1 μg of gC2, gD2, or gE2 - modified mRNA was transfected into 293T cells using TransIT - mRNA (Mirus Bio LLC) for transfection. After 18 hours, the cells were harvested and extracts were prepared for Western blot. The mRNAs were designed to express the extracellular domains of gC2, gD2, and gE2 (labeled mRNA - ecto). As a control for the expected molecular weight, purified baculovirus proteins gC2, gD2, and gE2 that express the same amino acids as the mRNA construct (labeled Bac - ecto) were used (Figures 1A - C).

[0343] Conclusion: When introduced into mammalian cells, the modified mRNAs encoding the extracellular domains of HSV-2 gC2 (Figure 1A), gD2 (Figure 1B), and gE2 (Figure 1C) produced proteins of appropriate molecules that reacted with antibodies against glycoproteins in Western blot.

[0344] Example 3: ELISA antibody responses in subjects immunized with gD2 or trivalent modified mRNA vaccine ELISA endpoint titers were evaluated in sera collected 28 days after the first and second immunizations. The immunization groups were as follows: multivalent C (multivalent C mRNA / LNP 10 μg divided into 4 aliquots and administered at 4 separate sites) (control); gD2 (gD2 mRNA / LNP 10 μg divided into 4 aliquots and administered at 4 separate sites); trivalent-I (gC2 mRNA / LNP 10 μg, gD2 mRNA / LNP 10 μg, gE2 mRNA / LNP 10 μg, each divided into 2 aliquots and administered at 2 sites respectively); and trivalent-C (gC2b mRNA 10 μg and gD2 mRNA 10 μg and gE2 mRNA 10 μg combined in LNP, divided into 4 aliquots and administered at 4 separate sites).

[0345] Four animals were evaluated in each group. After the first immunization (marked as Roman numeral I; Figures 2A - C), high ELISA titers were obtained against each immunogen, and after the second immunization (marked as Roman numeral II; Figures 2A - C), the titers were boosted even higher. Immunization with the gD2 - modified mRNA vaccine selectively induced ELISA antibodies against very high titers of gD2 (Figure 2B), while immunization with the trivalent - modified mRNA vaccine produced ELISA antibodies against very high titers of gC2 (Figure 2A) and gD2 (Figure 2B), as well as ELISA antibodies against high titers of gE2 (Figure 2C). In all non - control groups, the second immunization significantly boosted the ELISA titers compared to the first. The difference between the titers in the second immunization and those in the first immunization was significant, p < 0.05 (t - test, comparing the antibody titers after the first and second immunizations).

[0346] Conclusion: The gD2 mRNA as well as the gC2, gD2, and gE2 mRNA immunogens induced very high titers of ELISA antibodies after the first immunization and significantly boosted them after the second immunization.

[0347] Example 4: Balanced T H 1 and T H 2 IgG isotypes T H 1 or T H 2 The ability of mRNA immunization to predominantly stimulate the T H 2) or IgG2a (T H1) It was tested by determining whether antibodies were produced. ELISA was performed on plates coated with all three antigens gC2, gD2, and gE2. Serum obtained after the first or second immunization was added to plates coated with the antigen, and IgG1 or IgG2a was detected using HRP anti-mouse IgG1 or IgG2a. The IgG1 (Figure 3A) and IgG2a (Figure 3B) titers were significantly increased after immunization with gD2 and trivalent modified mRNA vaccination. Furthermore, the IgG1 (Figure 3A) or IgG2a (Figure 3B) titers were significantly higher after the second modified mRNA immunization compared to the first, p < 0.05 (t-test).

[0348] Conclusion: The results showed that antibody titers were generated against both IgG1 and IgG2a isotypes, which indicated a balanced T H 1 and T H 2 response.

[0349] Example 5: High neutralizing antibody titers after modified mRNA immunization Twenty-eight days after the second immunization, serum was obtained and neutralizing antibody titers were determined using serial two-fold dilutions of serum starting at a 1:25 dilution and 10% human serum as a source of complement. Human serum was obtained from individual serum negatives for HSV-1 and HSV-2. Each of the modified mRNA groups was significantly different from the multivalent C control (p < 0.001; Figure 4). While each of the mRNA groups was not significantly different from each other, trivalent vaccination (trivalent-C) administered as a combined immunogen was performed as the best of the three mRNA groups (Figure 4).

[0350] Conclusion: Each of the modified mRNA groups produced very high titers of neutralizing antibodies in the presence of 10% human complement.

[0351] Example 6: CD4 + and CD8 + T cell responses Five animals of the trivalent-modified mRNA group (trivalent-I group) immunized with each glycoprotein mRNA at separate sites were euthanized 14 days after the second immunization. Spleen cells were prepared for T cell assay. The spleen cells were stimulated with glycoprotein subunit antigen prepared in baculovirus or 15 amino acid peptides containing 11 overlapping amino acids. CD4 + and CD8 + T cell responses are shown in Figures 5 and 6, respectively.

[0352] CD4 + T cells: The modified mRNA-expressed gC2, gD2, and gE2 subunit antigens each stimulated multifunctional CD4 + T cell responses (Figures 5A - 5B). Spleen cells recovered from immunized subjects and then stimulated with subunit antigen glycoprotein increased multifunctional CD4 + T cell responses (Figure 5A). Spleen cells recovered from immunized subjects and then stimulated with 15 amino acid overlapping peptides increased multifunctional CD4 + T cell responses and IFNγ responses (Figure 5B). CD8 + T cells: Only the gE peptide pool 2 stimulated a significant IFNγ CD8 + T cell response (Figure 6B).

[0353] Example 7: Survival, weight loss, and neurological signs after modified mRNA immunization and intravaginal challenge Thirty-three days after the second immunization, the animals were challenged with 5×10 3 PFU of HSV-2 strain MS (about 400 LD 50They were inoculated intravaginally. The animals were observed daily for survival, neurological signs consisting of hindlimb weakness or paralysis and a hunched gait, and weight loss or gain. All animals in the polyvalent C control group died, while all animals in gD2 alone, the trivalent administered individually (labeled trivalent-I), or the trivalent administered in combination (labeled trivalent-C) survived (Figure 7A; p = 0.002 by Log-rank (Mantel-Cox) compared to the three mRNA / LNP groups using the polyvalent C control). Figure 7B shows that administration of the modified mRNA vaccine twice at 28-day intervals and intravaginal challenge with HSV-2 did not result in neurological signs or weight loss. Control subjects vaccinated and intravaginally challenged with HSV-2 showed weight loss and neurological signs.

[0354] Each of the mRNA / LNP groups was significantly superior to the control group. All mice immunized with the modified mRNA survived, showed no evidence of weight loss, neurological disease, or genital lesions after intravaginal challenge with approximately 400 LD 50 of HSV-2.

[0355] Example 8: HSV-2 vaginal titers after modified mRNA immunization and intravaginal challenge At 2 and 4 days after challenge, vaginal swabs were obtained from 10 animals per group and cultured for replication-competent HSV-2 virus. The results are shown in Figure 8. Nine out of 10 animals in the polyvalent C group had positive cultures at day 2 (Figure 8A) and day 4 (Figure 8B) compared to 3 out of 10 in the gD2 group and 0 out of 10 in the trivalent-I or trivalent-C groups (P values by Fisher Exact test were not significant when comparing the trivalent groups to gD2 alone; p < 0.001 when comparing trivalent-I or trivalent-C to polyvalent C; p = 0.02 when comparing gD2 alone to polyvalent C).

[0356] Each of the mRNA / LNP groups was significantly superior to the multivalent C control group. Notably, the vaginal titers on days 2 and 4 post-loading were negative in mice immunized with the trivalent mRNA, regardless of whether administered at separate sites or as combined immunizations. No significant difference was detected when comparing any of the trivalent groups to gD2 alone, but since 3 out of 10 mice in the gD2 group had virus isolated from vaginal swabs, both trivalent groups were superior to the gD2-alone group.

[0357] Example 9: Modified mRNA Immunization and Genital Disease after Vaginal Loading Animals were monitored daily for genital disease for 28 days post-loading. A score of 0 was designated as no disease, and 1 point each was designated for hair loss around the anus or genital opening, genital erythema, genital exudate, and necrosis of genital tissue (Figure 9).

[0358] Animals in the gD2 or trivalent mRNA / LNP groups did not develop genital disease and were significantly different from the multivalent C control (p < 0.001, one-way ANOVA by Kruskal-Wallis test followed by Dunn's multiple comparisons for significance).

[0359] Example 10: Modified mRNA Immunization and HSV-2 DNA in the Dorsal Root Ganglia after Vaginal Loading With the exception of the trivalent combined group in which 4 animals were euthanized, 5 animals per group were euthanized 4 days after loading. Dorsal root ganglia (DRG) were harvested for quantification of HSV-2 DNA by qPCR for detection of the Us9 gene. All 5 animals in the multivalent C group had HSV-2 DNA detected in the DRG, while 2 out of 5 animals in the gD mRNA, 1 out of 5 animals in the trivalent mRNA at separate sites, and 1 out of 4 in the trivalent mRNA administered at the same site were positive for HSV-2 DNA (Figure 10; Mann-Whitney test: gD2 compared to multivalent C, p = 0.03; trivalent at different sites compared to multivalent C, p < 0.01; trivalent at the same site compared to multivalent C, p = 0.14). The differences between the groups immunized with the modified mRNA were not significant.

[0360] Conclusion: In 75% - 80% of the animals immunized with gD2 alone or the trivalent vaccine, on the 4th day after infection, the dorsal root ganglia were positive for HSV-2 DNA. The trivalent mRNA groups and the gD2 mRNA group at different sites were significantly superior to the polyvalent C mRNA control group. On the other hand, the trivalent mRNA group containing all glycoproteins administered together was not significantly different from the polyvalent C group, probably due to the smaller sample size in the trivalent combined group.

[0361] Overview gD2 alone or the modified mRNA vaccine expressing gC2, gD2, and gE2 provided outstanding protection against HSV-2 genital load. The expression of the three proteins was significantly superior to that of gD2 based on the titers on the 2nd and 4th days after the load, and fewer numbers of animals using HSV-2 DNA were detected in the DRG on the 4th day.

[0362] Example 11: Follicular Helper T (Tfh) Cells and Germinal Center B Cell Responses in Immunized Mice BALB / c female mice were left non-immunized as negative control animals or were immunized intradermally with polyvalent C mRNA-LNP or trivalent modified mRNA-LNP at 28-day intervals. The polyvalent C mRNA control received 10 μg of polyvalent C mRNA-LNP administered at four separate sites in four aliquots. The trivalent modified mRNA groups each received 10 μg of gC2 mRNA-LNP, 10 μg of gD2 mRNA-LNP, and 10 μg of gE2 mRNA-LNP, each divided into two aliquots and administered at two sites respectively. Two weeks after the second immunization, spleens were harvested from five animals per group and flow cytometry was performed to detect follicular helper T (Tfh) cells (Figure 11A; * p < 0.05) and germinal center B cell responses (Figure 11B; * p < 0.05).

[0363] Conclusion: The trivalent mRNA-LNP vaccine induced strong Tfh and germinal center B cell responses, which were significantly superior to those of the multivalent C control immunization (p<0.05) and the negative group (p<0.05) for both Tfh and germinal center B cell responses. These immune responses suggest that the trivalent modified mRNA-LNP vaccine may induce a persistent antibody response.

[0364] Example 12: Vaginal IgG Response to Modified mRNA Immunization in Mice BALB / c mice were immunized intradermally twice at 28-day intervals with 10 μg of multivalent C mRNA-LNP, 10 μg of gD2 mRNA-LNP, or 10 μg each of gC2, gD2, and gE trivalent modified mRNA-LNP. The trivalent mRNA was combined, formulated with LNP, divided into four aliquots, and administered at four sites with 10 μg of gC2 mRNA, 10 μg of gD2 mRNA, and 10 μg of gE2 mRNA. One month after the second immunization, 60 μl of medium was introduced into the vaginal cavity and recovered. IgG titers against gC2 (Figure 12A), gD2 (Figure 12B), and gE2 (Figure 12C) were determined by ELISA in a 1:50 dilution of vaginal washings (Figure 12A-C, n = 10 mice in the multivalent C group, n = 10 in the gD2 mRNA group, and n = 25 in the trivalent mRNA group; *** p<0.001; ** p<0.01).

[0365] Conclusion: The trivalent mRNA induced a strong vaginal IgG response against gC2 (Figure 12A) and gD2 (Figure 12B), and a more moderate response against gE2 (Figure 12C). The gD2 ELISA titer was higher in mice immunized with the modified trivalent mRNA vaccine compared to mice immunized with the modified gD2 mRNA vaccine (Figure 12B).

[0366] Example 13: Antibodies against gC2 Produced by Trivalent mRNA Immunization of Mice Block the Immune Evasion Domain on gC2 BALB / c mice were left non-immunized as a source of non-immune IgG or were immunized intradermally with multivalent CmRNA-LNP or trivalent mRNA-LNP. The multivalent CmRNA control received 10 μg of multivalent CmRNA-LNP administered at four separate sites in four aliquots. The gD2mRNA group received 10 μg of gD2mRNA-LNP administered as described for multivalent CmRNA-LNP. The trivalent modified mRNA group received 10 μg of gC2mRNA-LNP, 10 μg of gD2mRNA-LNP, and 10 μg of gE2mRNA-LNP combined in one LNP and administered at four sites in four aliquots. There were 10 mice in each group. Serum from 10 mice was pooled and IgG was purified. IgG was evaluated for its ability to block complement component C3b bound to gC2 at 12 μg / 200 μl. This blocking assay was used to evaluate whether antibodies produced by immunization blocked the immune evasion properties of gC2. Non-immune mouse IgG, IgG from the multivalent CmRNA group, and IgG from the gD2mRNA group did not block gC2 bound to C3b, respectively. In contrast, IgG from mice immunized with trivalent mRNA blocked the interaction between gC2 and C3b overall (Figure 13, **** p < 0.0001).

[0367] Conclusion: The trivalent mRNA vaccine produces antibodies that block the immune evasion domain on gC2 as determined by blocking the interaction between gC2 and C3b.

[0368] Example 14: Higher Titer HSV-2 Vaginal Infection of Mice after Modified mRNA Vaccination BALB / c mice (n = 5) were immunized with trivalent modified mRNA using 10 μg of gC2mRNA-LNP, 10 μg of gD2mRNA-LNP, and 10 μg of gE2mRNA-LNP administered individually at two sites each in two aliquots. One month after the second immunization, the mice were treated with medroxyprogesterone and 5 days later, 5 × 104 Female mice were intravaginally infected with the PFU HSV-2 strain MS (2,000 LD 50 50). The animals were followed for 28 days, and at 2 and 4 days after infection, death, genital disease, and vaginal virus titers were evaluated. At 28 days after infection, the HSV-2 DNA copy number in the dorsal root ganglia (DRG) was evaluated. Mice immunized with the trivalent mRNA-LNP vaccine did not die, did not have genital disease, did not have virus detected at 2 or 4 days after infection, or did not have HSV-2 DNA detected in the DRG (Table 1).

Table 1

[0369] Conclusion: Mice were infected with 10-fold higher doses of HSV-2 than those used in the previous experiments described herein (Figures 7-10). Mouse protection remained outstanding even at this higher titer load. The inventors achieved stable immunity in all 5 mice as determined by no death, no genital disease, negative vaginal virus titers at 2 and 4 days after infection, and no HSV-2 DNA in the lumbosacral DRG at day 28 (Table 1).

[0370] Example 15: Evaluation of the Intramuscular Route of Modified mRNA Immunization in Mice BALB / c mice were immunized intramuscularly as controls with multivalent C mRNA-LNP (15 / group) or trivalent mRNA containing 10 μg each of gC2, gD2, and gE2 mRNA-LNP (20 / group). All multivalent C control animals died by day 12, while all animals in the trivalent mRNA group survived (Figure 14A). There was no weight loss in the trivalent mRNA group, while multivalent C control animals lost >15% of their body weight (Figure 14B). The multivalent C group developed extensive genital disease, while animals in the trivalent mRNA group had no genital disease (Figure 14C). DRGs were harvested at the time of euthanasia on days 7–12 post-infection from 9 multivalent C animals or at the end of the experiment on day 28 in the trivalent mRNA group. All animals in the multivalent C group had HSV-2 DNA detected in the DRG, while none were positive for HSV-2 DNA in the trivalent mRNA group (Figure 14D). Vaginal virus cultures on day 2 (Figure 14E) and day 4 (Figure 14F) were positive in all 15 animals in the multivalent C group, while the cultures were negative in all 20 animals in the trivalent mRNA group. The differences between the multivalent C and trivalent groups were significant, with p < 0.001 for each figure (Figures 14A–14F).

[0371] Conclusion: Trivalent modified mRNA-LNP confers outstanding protection in mice when administered intramuscularly. The inventors reported comparable findings above when immunizing mice intradermally. Overall, the inventors herein evaluated 64 mice immunized with 10 μg of each immunogen trivalent mRNA administered either intradermally (Figures 7–10) or intramuscularly (Figure 14). The inventors achieved stabilized immunity in 63 / 64 (98%) mice based on no deaths, no genital disease, no weight loss, negative vaginal titers on days 2 and 4, and negative HSV-2 DNA in the DRG.

[0372] Example 16: Overview of Comparison of Immunization with Trivalent mRNA-LNP and Trivalent Subunit Antigen CPG / Alum in BALB / c Mice The results presented in Table 2 below in this specification represent an overview of all results in BALB / c mice immunized either intradermally or intramuscularly with a trivalent mRNA containing 10 μg each of gC2, gD2, and gE2 mRNA-LNP (a total of 64 mice were studied). The inventors show a comparison with the results obtained in BALB / c mice immunized with 5 μg each of bac-gC2(27-426t) containing gC2 amino acids 27-426 from HSV-2 strain 333, bac-gD2(306t) containing gD2 amino acids 26-331 from HSV-1 strain 333, and bac-gE2(24-405t) containing gE2 amino acids 24-405 from HSV-2 strain 2.12. The gC2, gD2, and gE2 subunit antigens were mixed with 150 μg of CpG and 25 μg of alum per 1 μg of protein as an adjuvant and administered intramuscularly. As the inventors did in the previous experiment, the mice were immunized twice with the trivalent mRNA-LNP at 28-day intervals and three times with the subunit antigen at 14-day intervals. The mRNA and subunit antigen experiments were conducted simultaneously. The results outlined in Table 2 show a significant superiority of the trivalent mRNA-LNP vaccine over the trivalent subunit antigen vaccine in many immune response parameters and, most importantly, in vaccine efficacy. The trivalent mRNA-LNP vaccine achieved stabilized immunity in 63 / 64 (98%) mice compared to 15 / 20 (75%) in the subunit antigen group.

Table 2-1

Table 2-2

[0373] Example 17: Evaluation of a Trivalent MRNA-LNP Vaccine in Guinea Pigs Hartley female guinea pigs were left non-immunized and uninfected (negative group, n = 10) and were intradermally immunized three times at one-month intervals with 20 μg of multivalent C mRNA-LNP (n = 10) or 20 μg each of mRNA-LNP modified with gC2, gD2, and gE (n = 10). One month after the final immunization, animals in the multivalent C and trivalent mRNA groups were intravaginally infected with 5 × 10 5 PFU of the HSV-2 strain MS (50 LD 50 50). Animals were observed for death, genital lesions during the acute phase of infection (days 1 - 14), and genital lesions during the recurrence phase of infection (days 15 - 60). In the multivalent C control group, 7 out of 10 animals died or were humanely euthanized 7 - 20 days after infection, while none of the animals in the trivalent group and the untreated (non-infected) animals died (Figure 15A). The multivalent C group had genital lesions on the median day 6.4 during the acute phase of infection, including 9 out of 10 animals that developed acute genital disease, while none of the animals in the trivalent group or the untreated (non-infected) group developed acute genital disease (Figure 15B). The multivalent C animals had genital lesions on the median day 3.7 during the recurrence phase of infection, including 2 out of 3 animals that developed recurrent genital lesions (Figure 15C). In contrast, the trivalent-immunized guinea pigs and the untreated (non-infected) animals did not have recurrent genital lesions (Figure 15C). Conclusion: Trivalent-modified mRNA-LNP provided outstanding protection against acute and recurrent genital lesions in guinea pigs.

[0374] Preferred embodiments of the present invention are described with reference to the accompanying drawings, it being understood that the present invention is not limited to the exact embodiments and that various changes and modifications can be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0375] All patent documents and references cited herein are incorporated by reference as fully described. The present invention provides, for example, the following items. (Item 1) A composition comprising one or more nucleoside-modified mRNAs, wherein each of the nucleoside-modified mRNAs encodes a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and wherein the nucleoside-modified mRNA comprises one or more pseudouridine residues. (Item 2) The composition according to item 1, wherein the one or more pseudouridine residues comprise m1Ψ (1-methylpseudouridine). (Item 3) The one or more pseudouridine residues are 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-methylpseudouridine), or any combination thereof, of the composition according to item 1. (Item 4) The composition according to any one of items 1 to 3, wherein one or more of the nucleoside-modified mRNAs encode a) herpes simplex virus glycoprotein D (gD) or an immunogenic fragment thereof, b) herpes simplex virus glycoprotein C (gC) or an immunogenic fragment thereof, and c) herpes simplex virus glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof. (Item 5) The composition according to item 4, wherein the HSV glycoprotein comprises an HSV-1 glycoprotein. (Item 6) The composition according to item 4, wherein the HSV glycoprotein comprises an HSV-2 glycoprotein. (Item 7) The composition according to item 6, wherein the nucleoside-modified mRNA encoding the immunogenic fragment of the gD immunogenic fragment of HSV comprises amino acids 26 to 331 from the HSV-2 333 strain or a homologous sequence from another HSV strain. (Item 8) The composition according to item 7, wherein the nucleic acid sequence of the nucleoside-modified mRNA is as set forth in SEQ ID NO: 4. (Item 9) The immunogenic fragment of HSV gC comprises any one of its C3b binding domain, its properdin interference domain, its C5 interference domain, or a fragment of the C3b binding domain, properdin interference domain, or C5 interference domain, and is any one of items 4 to 8 The composition according to any one of the preceding paragraphs. (Item 10) The nucleoside-modified mRNA encoding the immunogenic fragment of HSV gC comprises amino acids 27 to 426 from HSV-2 strain 333 or a homologous sequence from another HSV strain, and is the composition according to any one of items 6 to 9. (Item 11) The nucleic acid sequence of the nucleoside-modified mRNA is as set forth in SEQ ID NO: 10, and is the composition according to item 10. (Item 12) The immunogenic fragment of HSV gE comprises amino acids 24 to 405 from HSV-2 strain 2.12 or a homologous sequence from another HSV strain, and is the composition according to any one of items 6 to 11. (Item 13) The nucleic acid sequence of the nucleoside-modified mRNA is as set forth in SEQ ID NO: 16, and is the composition according to item 12. (Item 14) One or more of the nucleoside-modified mRNAs encode a) HSV glycoprotein B (gB) or an immunogenic fragment thereof, b) HSV glycoprotein H (gH) or an immunogenic fragment thereof, c) HSV glycoprotein L (gL) or an immunogenic fragment thereof, d) HSV glycoprotein I (gI) or an immunogenic fragment thereof, or e) any combination thereof, and is the composition according to any one of items 1 to 13. (Item 15) One or more of the nucleoside-modified mRNAs further comprise a polyA tail, and is the composition according to any one of items 1 to 14. (Item 16) One or more of the nucleoside-modified mRNAs further comprise an m7GpppG cap, a 3'-O-methyl-m7GpppG cap, or an anti-reverse cap analog, and is the composition according to any one of items 1 to 15. (Item 17) The composition according to any one of Items 1 to 16, wherein one or more of the nucleoside-modified mRNAs further comprise a cap-independent translation enhancer. (Item 18) The composition according to any one of Items 1 to 17, wherein one or more of the nucleoside-modified mRNAs further comprise 5' and 3' untranslated regions that promote translation. (Item 19) The composition according to any one of Items 1 to 18, wherein one or more of the nucleoside-modified mRNAs are encapsulated in nanoparticles, lipids, polymers, cholesterol, or cell-penetrating peptides. (Item 20) The composition according to Item 19, wherein the nanoparticles are liposomal nanoparticles. (Item 21) A method for treating herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition of a nucleoside-modified mRNA according to any one of Items 1 to 20. (Item 22) A method for suppressing, inhibiting, or reducing the occurrence of herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition of a nucleoside-modified mRNA according to any one of Items 1 to 20. (Item 23) The method according to any one of Items 21 to 22, wherein the HSV infection includes HSV-1 infection. Method. (Item 24) The method according to any one of Items 21 to 22, wherein the HSV infection includes HSV-2 infection. (Item 25) The method according to any one of Items 21 to 24, wherein the HSV infection includes primary HSV infection. (Item 26) The method according to any one of Items 21 to 24, wherein the HSV infection includes relapse, recurrence, or oral HSV following primary HSV infection. (Item 27) The HSV infection is the method according to any one of items 21 to 24, including reactivation of latent HSV infection. (Item 28) The HSV infection is the method according to any one of items 21 to 27, including HSV encephalitis, HSV neonatal infection, genital HSV infection, or oral HSV infection. (Item 29) A method for inducing an immune response in a subject, comprising administering to the subject a composition of nucleoside-modified mRNA according to any one of items 1 to 20. (Item 30) The administration step is the method according to any one of items 21 to 29, including intramuscular administration. (Item 31) The administration step is the method according to any one of items 21 to 29, including subcutaneous administration. (Item 32) The administration step is the method according to any one of items 21 to 29, including intradermal administration. (Item 33) The administration step is the method according to any one of items 21 to 29, including intranasal, intravaginal, or rectal administration. (Item 34) The administration step is the method according to any one of items 21 to 29, including topical administration. (Item 35) The administration step includes a) administering a first composition comprising nucleoside-modified mRNA encoding a first HSV glycoprotein, b) administering a second composition comprising nucleoside-modified mRNA encoding a second HSV glycoprotein, and c) administering a third composition comprising nucleoside-modified mRNA encoding a third HSV glycoprotein, and is the method according to any one of items 21 to 34. (Item 36) The first composition, the second composition, and the third composition are administered to the subject at a single administration site, according to the method of item 35. (Item 37) The method according to item 35, wherein the first composition, the second composition, and the third composition are administered to the subject at different administration sites. (Item 38) The method according to any one of items 35 to 37, wherein the first composition, the second composition, and the third composition are administered simultaneously. (Item 39) The method according to any one of items 35 to 37, wherein the first composition, the second composition, and the third composition are administered sequentially. (Item 40) The first composition, the second composition, and the third composition are administered by the same administration route, according to the method of any one of items 35 to 39. (Item 41) The method according to any one of items 35 to 39, wherein the first composition, the second composition, and the third composition are administered by different administration routes. (Item 42) The method according to any one of items 21 to 41, further comprising administering to the subject one or more additional administrations of the composition of the nucleoside-modified mRNA after the first administration. (Item 43) The method according to any one of items 21 to 41, further comprising administering to the subject a composition comprising the HSV glycoprotein or an immunogenic fragment thereof. (Item 44) The method according to item 43, wherein the composition comprising the HSV glycoprotein is administered after the administration of the composition of the nucleoside-modified mRNA. (Item 45) The method according to item 43, wherein the composition comprising the HSV glycoprotein is administered before the administration of the composition of the nucleoside-modified mRNA. (Item 46) The method according to any one of items 43 to 45, wherein the HSV glycoprotein comprises gD, gC, gE, or a combination thereof. (Item 47) The immune response is the method according to any one of items 29 to 46, including a CD4 immune response. (Item 48) The immune response is the method according to any one of items 29 to 46, including a CD8 immune response. (Item 49) The immune response is the method according to any one of items 29 to 46, including a follicular helper T cell immune response. (Item 50) The immune response is the method according to any one of items 29 to 46, including a germinal center B cell immune response. (Item 51) The immune response is the method according to any one of items 29 to 46, including an IgG antibody response against gC2, gD2, gE2, or a combination thereof.

Claims

1. 1. Use of a composition for the manufacture of a medicament for suppressing, inhibiting, or reducing the incidence of herpes simplex virus (HSV) infection in a subject, said composition comprising: (i) RNA encoding an immunogenic fragment of HSV glycoprotein D (gD); (ii) RNA encoding an immunogenic fragment of HSV glycoprotein C (gC), and (iii) RNA encoding an immunogenic fragment of HSV glycoprotein E (gE). wherein one or more of the RNAs is a nucleoside-modified RNA.

2. 1. Use of a composition for the manufacture of a medicament for inducing an immune response to herpes simplex virus (HSV) infection in a subject, said composition comprising: (i) RNA encoding an immunogenic fragment of HSV glycoprotein D (gD); (ii) RNA encoding an immunogenic fragment of HSV glycoprotein C (gC), and (iii) RNA encoding an immunogenic fragment of HSV glycoprotein E (gE). wherein one or more of the RNAs is a nucleoside-modified RNA.

3. The use according to claim 1 or 2, wherein at least one of the nucleoside-modified RNAs comprises one or more pseudouridine residues.

4. The one or more pseudouridine residues are １ Ψ (1-methylpseudouridine), 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-methylpseudouridine), or any combination thereof.

5. The composition comprises: (a) RNA encoding an immunogenic fragment of HSV glycoprotein B (gB); (b) RNA encoding an immunogenic fragment of HSV glycoprotein H (gH); (c) RNA encoding an immunogenic fragment of HSV glycoprotein L (gL), and / or (d) RNA encoding an immunogenic fragment of HSV glycoprotein I (gI).

3. The use according to claim 1 or 2, further comprising:

6. The use according to claim 1 or 2, wherein one or more of the immunogenic fragments of HSV gC, HSV gD and HSV gE are derived from HSV-1.

7. The use according to claim 1 or 2, wherein one or more of the immunogenic fragments of HSV gC, HSV gD and HSV gE are derived from HSV-2.

8. One or more of the RNAs (a) a polyA tail; (b) m7GpppG cap, 3'-O-methyl-m7GpppG cap, or anti-reverse cap analog; (c) a cap-independent translation enhancer, and / or (d) 5' and 3' untranslated regions that facilitate translation 3. The use according to claim 1 or 2, further comprising:

9. 3. The use according to claim 1 or 2, wherein one or more of the RNAs are encapsulated in a nanoparticle, a lipid, a polymer, cholesterol, or a cell-penetrating peptide.

10. 10. The use according to claim 9, wherein the one or more of the nucleoside-modified RNAs are encapsulated in a nanoparticle, the nanoparticle being a liposomal nanoparticle.

11. The use according to claim 1 or 2, wherein one or more of the nucleoside-modified RNAs further comprises a signal sequence.

12. 3. The use of claim 1 or 2, wherein the RNA encoding an immunogenic fragment of HSV gD is nucleoside-modified RNA, the RNA encoding an immunogenic fragment of HSV gC is nucleoside-modified RNA, and the RNA encoding an immunogenic fragment of HSV gE is nucleoside-modified RNA.

13. The use according to claim 1 or 2, wherein the HSV infection comprises an HSV-1 infection.

14. The use according to claim 1 or 2, wherein the HSV infection comprises an HSV-2 infection.

15. 3. The use of claim 1 or 2, wherein the HSV infection comprises a primary HSV infection, a flare-up, recurrence or oral HSV following a primary HSV infection, a reactivation of a latent HSV infection, a flare-up or recurrence following reactivation of a latent HSV infection, HSV encephalitis, a neonatal HSV infection, a neonatal HSV encephalitis infection, a genital HSV infection, or an oral HSV infection, and optionally the primary HSV infection or the reactivation of the latent HSV infection comprises the HSV encephalitis, the neonatal HSV infection, the neonatal HSV encephalitis infection, the genital HSV infection, or the oral HSV infection.

16. The use of claim 2 , wherein the immune response comprises a CD4 immune response, a CD8 immune response, a follicular helper T cell immune response, a germinal center B cell immune response, or an IgG antibody response against HSV gC2, HSV gD2 and / or HSV gE2.

17. 3. The use according to claim 1 or 2, wherein the medicament is formulated for intramuscular, subcutaneous, intradermal, intranasal, intravaginal, intrarectal or topical administration.

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