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 compositions and methods of use for the prevention and treatment of genital herpes, comprising nucleoside-modified mRNA encoding herpes simplex virus (HSV) glycoproteins, including those involved in viral entry and immune evasion. [Background technology]

[0002] A genital herpes vaccine is urgently needed to prevent pain and suffering, reduce the incidence of neonatal herpes, and lower the risk of HIV acquisition and transmission that occurs with genital infection. 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 quiescent. In both circumstances, 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 is currently available.

[0003] Chiron Corp. investigated a prophylactic vaccine containing glycoproteins B (gB2) and D (gD2), two HSV-2 glycoproteins involved in viral entry, given with MF59 as an adjuvant. The vaccine delayed the onset of infection over the first 5 months after immunization, but seronegative partners were not protected from HSV-2 infection. GlaxoSmithKline (GSK) evaluated a prophylactic vaccine using the gD2 antigen with monophosphoryl lipid A (MPL) and alum as adjuvants. Overall, significant protection was observed in a subgroup of women who were doubly seronegative for HSV-1 and HSV-2, but no protection against genital lesions was detected. Follow-up studies were performed in doubly seronegative women in whom there was no overall protection against genital herpes; however, the vaccine was effective against HSV-1. This result was noteworthy because HSV-1 accounted for 60% of genital herpes infections in the control group, suggesting that these studies were not sufficient to target a vaccine that blocks HSV-2 entry.

[0004] HSV-1 and HSV-2 gC 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, which activates the membrane attack complex leading to virus neutralization and lysis of infected cells. C3b stimulates B- and T-cell responses and serves as a link between innate and adaptive immunity. HSV-1 and HSV-2 gC bind C3b and inhibit C3b-mediated activity. 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 the Fc domain of an IgG molecule that binds to its target through its F(ab')2 domain. Vaccines containing gE2 subunit antigens produce antibodies that bind to gE2 and block its immune evasion function. HSV-2 gC2 and gE2 perform activities similar to mammalian complement and IgG Fc regulatory proteins, but share no sequence homology with mammalian receptors, effectively suggesting that there is no risk that immunization will induce autoimmunity.

[0006] Previous studies from our laboratory have tested vaccines containing gC, gD, and gE and found that such vaccines provided protection against HSV infection, however, it is not known whether an mRNA vaccine encoding HSV gC, gD, and gE will be effective in protecting against HSV infection.

[0007] There are several advantages to using nucleic acids as vaccines: they can induce both humoral and cellular immune responses; they have low effective doses; they are easy to handle; they lend themselves to rapid testing; they are cost-effective and reproducible in terms of large-scale production and isolation; they can be produced frequently and are easy to isolate; they are more temperature stable than traditional vaccines; they have a longer shelf life; they are easier to store and transport; and they do not appear to require a cold chain (Shedlock & Weiner, J Leukocyte Biol. Vol 68, 2000).

[0008] In principle, either exogenous DNA or RNA can express proteins in the mammalian body. It is unclear whether it is possible to produce similar immune activity using both DNA and mRNA expressed proteins. It is generally accepted that DNA 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 fairly stable and non-reactive, and can be stored for long periods of time. However, DNA is self-replicating and can be easily damaged by ultraviolet radiation. DNA-based vaccines can also raise safety concerns due to possible insertion of DNA into genomes, possible interruption of genes, and formation of anti-DNA antibodies.

[0010] RNA vaccines present important safety features. RNA is more reactive and unstable than DNA, but is resistant to UV radiation. mRNA does not integrate 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 increases its safety. mRNA is more efficient than DNA for protein production in postmitotic, non-dividing cells because DNA requires translocation through nuclear members and the plasma membrane, whereas mRNA requires translocation only through the plasma membrane. mRNA is not only a template for translation, but also acts as a ligand for Toll-like receptors and is nuclease sensitive; therefore, there is less concern about horizontal transmission. Moreover, RNA vaccines elegantly incorporate adjuvant activity and antigen expression, thereby mimicking the relevant aspects of 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 address a range of specialized immune pattern recognition receptors, including Toll-like receptors 3, 7, and 8, RIG-I, MDA5, PKR, and other receptors that may act synergistically to enhance the induction of antigen-specific adaptive B and T cell responses. Importantly, by synthesising antigens in transfected host cells, mRNA vaccines directly introduce antigens into the cellular antigen processing and presentation pathway, establishing access to MHC molecules to induce T cell responses regardless of host MHC haplotype. This allows the induction of polyclonal T cell responses that can act synergistically with other immune responses, including B cells. Endogenous antigen production also ensures faithful post-translational modifications (eg, protein processing, glycosylation, etc.) that can positively influence immunogenicity. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Shedlock & Weiner, J Leukocyte Biol. Vol 68, 2000 Summary of the Invention [Means for solving the problem]

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

[0013] In another embodiment, the present invention provides a composition comprising one or more nucleoside modified mRNAs, each of which encodes a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and wherein the nucleoside modified mRNAs comprise 1-methylpseudouridine, wherein 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 invention provides a composition comprising modified mRNAs comprising one or more pseudouridine residues, each of which encodes a) HSV glycoprotein D (gD) or an immunogenic fragment thereof, b) HSV glycoprotein C (gC) or an immunogenic fragment thereof, c) HSV glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof.

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

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

[0018] In yet another embodiment, the present invention provides a method of treating a herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition comprising one or more modified mRNAs encoding a) HSV glycoprotein D (gD) or an immunogenic fragment thereof, b) HSV glycoprotein C (gC) or an immunogenic fragment thereof, c) HSV 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 incidence of herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition comprising one to three modified mRNAs, each of which encodes a) HSV glycoprotein D (gD) or an immunogenic fragment thereof, b) HSV glycoprotein C (gC) or an immunogenic fragment thereof, and c) HSV 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 following detailed description of the invention, examples and drawings. However, it should be understood that the detailed description of the invention and specific examples thereof, while showing preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description of the invention. [Brief description of the drawings]

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or application publication including color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] [Figure 1] 1A to 1C. Characterization of post-translational products of gC2-, gD2-, and gE2-modified mRNA ectodomains in Vero cells.

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

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

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

[0026] [Diagram 2] Figure 2A shows gC2 antibody (Ab) responses determined by antigen-specific ELISA in mice immunized with gD2 mRNA; or gC2 mRNA, gD2 mRNA, and gE2 mRNA, each given at different intradermal sites (trivalent-I); or gC2 mRNA, gD2 mRNA, and gE2 mRNA given in combination (trivalent-C). I indicates the first immunization; II indicates the second immunization.

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

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

[0029] [Diagram 3] Figure 3A shows antigen-specific IgG1 responses in mRNA-vaccinated mice. For IgG1 responses, antibodies were evaluated after the first and second immunizations. I indicates the first immunization; II indicates the second immunization.

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

[0031] [Figure 4]Figure 4: Neutralizing antibody titers in mRNA vaccinated mice. After the second immunization, 50% endpoint neutralizing titers of serum were obtained. Titers were performed with 10% human complement. Trivalent-I animals were immunized with gC2 / liposomal nanoparticles (LNP), gD2 / LNP, and gE2 / LNP given at different sites, respectively. Trivalent-C animals were immunized with gC2, gD2, and gE2 combined into a single LNP. P values ​​comparing 50% endpoint neutralizing titers were as follows: trivalent-I vs. gD2, p=0.04; trivalent-C vs. gD2, p=0.002; trivalent-I vs. trivalent-C, p-0.026.

[0032] [Diagram 5] Figure 5. CD4+ T cell responses to gC2, gD2 and gE2 mRNA, each administered at a different intradermal site. Splenocytes were stimulated with subunit antigen glycoproteins (Figure 5A) or with a 15 amino acid peptide with 11 overlapping amino acids that stimulates HSV-2 specific T cell responses (Figure 5B). * indicates p<0.05 (t test) comparing gC, gD or gE with PBS or DMSO stimulated CD4+ T cells. Error bars represent SD.

[0033] [Figure 6] Figure 6. CD8+ T cell responses to gC2, gD2 and gE2 mRNA, each administered at different intradermal sites. Splenocytes were stimulated with subunit antigen glycoproteins (Figure 6A) or with a 15 amino acid peptide with 11 overlapping amino acids that stimulates HSV-2 specific T cell responses (Figure B). * indicates p<0.05 comparing gE pool 2 with DMSO control. Error bars represent SD.

[0034] [Figure 7]Figure 7A shows the survival rate of BALB / c mice immunized with mRNA twice, 28 days apart, and challenged intravaginally with HSV-2. Trivalent-I represents animals immunized with gC2 / LNP, gD2 / LNP, and gE2 / LNP, each given at a different intradermal site. Trivalent-C represents animals immunized with gC2, gD2, and gE2 combined into a single LNP for immunization.

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

[0036] [Figure 8] Figures 8A to 8B are vaginal viral titers in mRNA vaccinated mice after intravaginal challenge with HSV-2. Vaginal swab titers were obtained on days 2 (Figure 8A) and 4 (Figure 8B) after challenge. The dotted line indicates the detection limit of the assay at 7 PFU / ml.

[0037] [Figure 9] Figure 9. Genital disease in mRNA vaccinated mice after HSV-2 intravaginal challenge. Mice were immunized with polyvalent C as a control, or with gD2 mRNA / LNP, trivalent for each glycoprotein mRNA at separate sites (trivalent-I) or trivalent combining all three mRNAs (trivalent-C). Genital disease was scored for 28 days. All animals in the polyvalent C group died by day 10. *** indicates p<0.001 comparing the polyvalent C group with the other three groups.

[0038] [Figure 10]Figure 10. HSV-2 DNA copy number in dorsal root ganglia (DRG) of mRNA vaccinated mice 4 days post-challenge. HSV-2 DNA in DRG was measured by qPCR. DRG from 4-5 animals per group were assessed for HSV-2 DNA 4 days post-challenge. Bars represent the mean value per group.

[0039] [Figure 11] In Figure 11A, trivalent mRNA-LNP vaccine induces strong follicular helper T cell responses in mice. BALB / c female mice were left unimmunized as naive control animals or were immunized intradermally twice with polyvalent C mRNA-LNP or trivalent modified mRNA-LNP at 28-day intervals. Polyvalent C mRNA control received 10 μg of polyvalent C mRNA-LNP divided into 4 aliquots and administered at 4 separate sites. 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 sites each. Two weeks after the second immunization, spleens were harvested from 5 animals per group and flow cytometry was performed to detect follicular helper T (Tfh) cell responses (*p<0.05).

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

[0041] [Figure 12] Figures 12A-C are genital mucosal IgG antibody responses. BALB / c mice were immunized intradermally twice with 10 μg of polyvalent C mRNA-LNP, 10 μg of gD2 mRNA-LNP, or 10 μg each of trivalent modified mRNA-LNP of gC2, gD2, and gE, with an interval of 28 days. Trivalent mRNA was administered in combination with LNP as 10 μg of gC2 mRNA, 10 μg of gD2 mRNA, and 10 μg of gE2 mRNA given at four sites in four aliquots. One month after the second immunization, 60 μl of medium was introduced into the vaginal cavity and collected. IgG titers were determined by ELISA against gC2 (FIG. 12A), gD2 (FIG. 12B), and gE2 (FIG. 12C) at a 1:50 dilution of vaginal washes (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).

[0042] [Figure 13] In FIG. 13, the trivalent mRNA-LNP vaccine produces antibodies that block gC2 from binding to complement component C3b. BALB / c mice were left unimmunized as a source of non-immune IgG or were immunized intradermally with polyvalent C mRNA-LNP or trivalent mRNA-LNP. The polyvalent C mRNA control received 10 μg of polyvalent C mRNA-LNP administered in four separate sites in four aliquots. The gD2 mRNA group received 10 μg of gD2 mRNA-LNP administered as described for polyvalent C mRNA-LNP. 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 given in four sites in four aliquots in line with LNP. Each group had 10 mice. Serum from the 10 mice was pooled and IgG was purified. This IgG was evaluated at 12 μg / 200 μl for its ability to block the binding of complement component C3b to gC2 (****p<0.0001).

[0043] [Figure 14-1] In Figures 14A to 14F, the trivalent mRNA vaccine provides significant protection in mice when the vaccine is administered intramuscularly. BALB / c mice were immunized intramuscularly with polyvalent C mRNA-LNP as a control (15 / group) or with trivalent mRNA containing 10 μg each of gC2, gD2 and gE2 mRNA-LNP (20 / group). Figure 14A represents mouse survival data; Figure 14B represents weight loss data; Figure 14C represents reproductive disease data. DRGs were harvested from nine polyvalent C animals at the time of euthanasia between days 7 and 12 post-infection or at the end of the experiment on day 28 in the trivalent mRNA group. Figure 14D represents HSV-2 DNA data from DRG. Figure 14E represents vaginal virus culture data on day 2 and Figure 14F represents vaginal virus culture data on day 4. The difference between the multivalent C group and the trivalent group was significant, p<0.001 in Figures 14A-14F. [Figure 14-2] In Figures 14A to 14F, the trivalent mRNA vaccine provides significant protection in mice when the vaccine is administered intramuscularly. BALB / c mice were immunized intramuscularly with polyvalent C mRNA-LNP as a control (15 / group) or with trivalent mRNA containing 10 μg each of gC2, gD2 and gE2 mRNA-LNP (20 / group). Figure 14A represents mouse survival data; Figure 14B represents weight loss data; Figure 14C represents reproductive disease data. DRGs were harvested from nine polyvalent C animals at the time of euthanasia between days 7 and 12 post-infection or at the end of the experiment on day 28 in the trivalent mRNA group. Figure 14D represents HSV-2 DNA data from DRG. Figure 14E represents vaginal virus culture data on day 2 and Figure 14F represents vaginal virus culture data on day 4. The difference between the multivalent C group and the trivalent group was significant, p<0.001 in Figures 14A-14F.

[0044] [Figure 15]In Figures 15A to 15C, the trivalent mRNA vaccine is highly effective in a guinea pig genital infection model. Hartley strain female guinea pigs were left unimmunized and uninfected (naive group, n=10) and were immunized intradermally three times at one-month intervals with 20 μg of polyvalent C mRNA-LNPs (n=10) or with 20 μg each of gC2, gD2, gE modified mRNA-LNPs (n=10). One month after the final immunization, animals in the polyvalent C and trivalent mRNA groups were infected intravaginally with 5 x 105 PFU of HSV-2 strain MS (50LD50). Animals were observed for death, genital lesions during the acute phase of infection (days 1-14), and genital lesions during the relapse phase of infection (days 15-60). FIG. 15A presents survival data; FIG. 15B provides data for vaginal disease (acute phase); and FIG. 15C provides data for vaginal disease (recurrent phase). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0046] In one embodiment, the invention provides a composition comprising one or more nucleoside modified mRNAs, each of which encodes a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and wherein the modified mRNA comprises one or more pseudouridine 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] Thus, in one embodiment, the invention provides a composition comprising one or more modified mRNAs encoding HSV gD, gC, gE, gB, gH, gL, gI, or immunogenic fragments thereof, hi one embodiment, the modified mRNA comprises a pseudouridine-modified mRNA.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0065] In another embodiment, the present invention provides a composition comprising (a) a modified mRNA encoding HSV-1 gD or a fragment thereof, (b) a modified mRNA encoding HSV-1 gC or a fragment thereof, and (c) a modified mRNA encoding HSV-1 gE 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 essentially 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 a gD protein of HSV, a modified mRNA encoding a gC protein of HSV, a modified mRNA encoding a gE protein of HSV, and a modified mRNA encoding one or more additional HSV glycoproteins. In one embodiment, the additional HSV glycoproteins comprise 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 glycoproteins comprise 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 of the invention and for use in the methods of the invention comprise both HSV-2 glycoproteins or glycoprotein fragments and HSV-1 glycoproteins or glycoprotein fragments. In another embodiment, the compositions of the invention and for use in the methods of the invention comprise a mixture of HSV-2 glycoproteins or glycoprotein fragments and HSV-1 glycoproteins or glycoprotein fragments. For example, in one embodiment, the compositions of the invention comprise HSV-2 gC, HSV-1 gD, and HSV-2 gE, or fragments thereof. In another embodiment, the compositions of the invention comprise HSV-1 gC, HSV-2 gD, and HSV-2 gE, or fragments thereof. In another embodiment, the compositions of the invention comprise HSV-2 gC, HSV-2 gD, and HSV-1 gE, or fragments thereof. In another embodiment, the compositions of the invention comprise HSV-1 gC, HSV-1 gD, and HSV-2 gE, or fragments thereof. In another embodiment, the composition of the invention comprises HSV-1 gC, HSV-2 gD, and HSV-1 gE, or fragments thereof. In another embodiment, the composition of the invention comprises HSV-2 gC, HSV-1 gD, and HSV-1 gE, or fragments thereof.

[0069] In another embodiment, the composition of the invention comprises one or more additional HSV-1 or HSV-2 glycoproteins, or both HSV-1 and HSV-2 glycoproteins, as described herein. For example, in one embodiment, a composition of the invention comprising HSV-2 gC, HSV-1 gD, and HSV-2 gE may further comprise HSV-1 gI. In another embodiment, a composition of the invention comprising HSV-2 gC, HSV-2 gD, and HSV-2 gE may further comprise HSV-1 gB. Each possible combination of HSV-1 and HSV-2 glycoproteins represents a separate embodiment of the present invention.

[0070] In one embodiment, "encoding" refers to an RNA molecule that contains a gene that encodes a protein of interest. In another embodiment, the RNA molecule comprises a protein coding sequence that encodes 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 a separate embodiment of the present invention.

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

[0072] In one embodiment, "immunogenicity" or "immunogenic" as used herein refers to the inherent ability of a protein, peptide, nucleic acid, antigen, or organism to elicit an immune response in an animal when the protein, peptide, nucleic acid, antigen, or organism is administered to the animal. Thus, in one embodiment, "promoting immunogenicity" refers to increasing the ability of a protein, peptide, nucleic acid, antigen, or organism to elicit an immune response in an animal when the protein, peptide, nucleic acid, antigen, or organism is administered to the animal. An increased ability of a protein, peptide, nucleic acid, antigen, or organism to elicit an immune response can, in one embodiment, be measured by an increased number of antibodies against the protein, peptide, nucleic acid, antigen, or organism, an increased diversity of antibodies against the antigen or organism, an increased number of T cells specific for the protein, peptide, nucleic acid, antigen, or organism, an increased cytotoxic or helper T cell response against the protein, peptide, nucleic acid, antigen, or organism, and the like.

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

[0074] In one embodiment, "function" within the meaning of this invention refers to the inherent ability of a protein, peptide, nucleic acid, fragment or variant thereof to exhibit a biological activity or function. In one embodiment, such biological function is its binding property to an interaction partner, e.g. a membrane-bound receptor, and in another embodiment, its trimerization property. In the case of functional fragments and variants of the invention, these biological functions may actually be altered, e.g. according to their specificity or selectivity, while the basic biological function is retained.

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

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

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

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

[0079] In one embodiment, compositions of modified mRNA encoding gD-1 are protective against HSV-1 infection. Also, combination compositions of modified mRNA encoding gC-1 / gD-1 / gE-1 provide superior protection compared to compositions containing modified mRNA encoding gC-1 alone, gD-1 alone, or gE-1 alone. Also, as provided herein, compositions of modified mRNA encoding gD-2 are protective against HSV-2 infection (Figures 7 to 10). Also, combination compositions of modified mRNA encoding gC-2 / gD-2 / gE-2 provide superior protection compared to compositions containing modified mRNA encoding gC-2 alone, gD-2 alone, or gE-2 alone.

[0080] In another embodiment, the inclusion of a modified mRNA encoding gC and / or a modified mRNA encoding gE in a composition of the invention increases the effectiveness of anti-gD antibodies elicited by the composition. In another embodiment, the inclusion of a modified mRNA encoding gC and / or a modified mRNA encoding gE in a composition of the invention increases the dose of modified mRNA encoding gD required to elicit antibodies that inhibit gD binding to a cellular receptor. In another embodiment, the inclusion of a modified mRNA encoding gC and / or a modified mRNA encoding gE in a composition of the invention reduces the dose of modified mRNA encoding gD required to elicit antibodies that inhibit gD binding to a cellular receptor when the modified mRNA encoding gD is administered separately from the modified mRNA encoding the gC or gE protein.

[0081] In another embodiment, the composition of the invention includes a modified mRNA encoding gC and / or a modified mRNA encoding gE to enhance 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 a NK (natural killer) cell response. In another embodiment, the innate immune response is any other innate immune response known in the art.

[0082] In another embodiment, the inclusion of a modified mRNA encoding gC and / or a modified mRNA encoding gE in a composition of the invention increases the effectiveness of antibodies elicited by the composition against one of the above glycoproteins. In another embodiment, the inclusion of a modified mRNA encoding gC and / or a modified mRNA encoding gE in a composition of the invention reduces the dose of one of the above glycoproteins required to elicit antibodies that inhibit the glycoprotein from binding to its cellular receptor when the one of the glycoproteins is administered separately from the other one of the glycoproteins. Glycoprotein D

[0083] In one embodiment, the composition of the 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 HSV-1 gD comprises: [ka] [ka] [ka]

[0085] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent 5' untranslated sequences. In one embodiment, the bolded residues represent a signal sequence (leader sequence) that aids in expression of the gD1 fragment. In one embodiment, the italicized residues represent 3' untranslated sequences 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 gD of HSV-1 encoded by the modified mRNA used in the methods and compositions of the invention includes amino acids 26-331 of gD from the Patton strain of HSV-1, set forth in the following amino acid sequence: KYALADASLKMADPNRFRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPY (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 invention comprises the following amino acid sequence: MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKLADPNRFRRKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYY DSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEAPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY (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 set forth 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 HSV-1 gD encoded by the modified mRNA used in the methods and compositions of the invention comprises an amino acid sequence set forth in any of the following GenBank Accession Nos.: 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, ABM66849.1, ABM66850.1, ABM66851.1, ABM66852.1, ABM66853.1, ABM66854.1, ABM66855.1, ABM66856.1, ABM66857.1, ABM66858.1, ABM66859 ...9.1, ABM66851.1, ABM66852.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853.1, ABM66853 CM62295.1, ADD60053.1, ADD60130.1, ADM22389.1, ADM22466.1, ADM22542.1, ADM22619.1, ADM22696.1, ADM22773.1, ADM22849.1, ADM22926.1, ADM2 3003.1, ADM23079.1, ADM23155.1, ADM23231.1, ADM23309.1, ADM23383.1, ADM23457.1, ADM23531.1, ADM23605.1, ADM23680.1, ADM23755.1, ADM2383 1.1, 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, AJE600 80.1, AJE60151.1, AJE60222.1, AJE60293.1, AJE60439.1, AKE48645.1, AK G59246.1, AKG59318.1, AKG59391.1, AKG59462.1, AKG59536.1, AKG59609.1, AKG59682.1, AKG59755.1, AKG59826.1, AKG59898.1, AKG59972.1, AKG60 046.1, AKG60118.1, AKG60189.1, AKG60261.1, AKG60334.1, AKG60404.1, AKG60474.1, AKG60546.1, AKG60620.1, AKG60692.1, AKG60763.1, AKG60835.<h2 style=";text-align:left;direction:ltr">1、AKG60906.1、AKG60978.1、AKG61050.1、AKG61123.1、AKG61194.1、AKG61 267.1、AKG61339.1、AKG61411.1、AKG61484.1、AKG61556.1、AKG61629.1、AK G61703.1, AKG61774.1, AKG61847.1, AKG61920.1, AKG61993.1, AKH80463.1, AKH80536.1, ALM22635.1, ALM22709.1, ALM22783.1, ALM22857.1, ALO186 62.1, 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、ANN846 53.1、ANN84730.1、ANN84806.1、ANN84883.1、ANN84959.1、ANN85036.1、ANN 85112.1、ANN85187.1、ANN85264.1、ANN85341.1、ANN85416.1、ANN85494.1 、ANN85571.1、ANN85648.1、ANN85724.1、ANN85801.1、AOY34093.1、AOY3414 1.1, 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, ARO3 7978.1, 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, A SM47741.1, 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, SB O07855.1, 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: [ka] [ka]

[0093] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent 5' untranslated sequences. In one embodiment, the bolded residues represent a signal sequence (leader sequence) that aids in expression of the gD2 fragment. In one embodiment, the italicized residues represent 3' untranslated sequences 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 sequence, a signal sequence, a 3' untranslated sequence, 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 the HSV-2 gD fragment set forth in the amino acid sequence Contains amino acids 26 to 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 invention comprises the following amino acid sequence: MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYY DSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY (sequence number 6).

[0097] In another embodiment, the HSV-2 gD encoded by the modified mRNA used in the methods and compositions of the invention comprises the amino acid sequence set forth in the following GenBank Accession Nos.: 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, AF S18201.1, AFS18202.1, AFS18203.1, AFS18204.1, AFS18205.1, AFS18206.1, AFS18207.1, AFS18208.1, AFS18209.1, AFS18210.1, AFS18211.1, AFS182 12.1, 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, AI L28069.1, AIL28070.1, AKC42828.1, AKC59305.1, AKC59376.1, AKC59447.1, AKC59518.1, AKC59589.1, AMB66102.1, AMB66171.1, AMB66244.1, AMB66 321.1, AMB66394.1, AMB66463.1, AQZ55754.1, AQZ55825.1, AQZ55896.1, A QZ55967.1, AQZ56038.1, AQZ56109.1, AQZ56180.1, AQZ56251.1, AQZ56322.<h2 style=";text-align:left;direction:ltr">1、AQZ56393.1、AQZ56464.1、AQZ56535.1、AQZ56606.1、AQZ56677.1、AQZ56 748.1、AQZ56819.1、AQZ56890.1、AQZ56961.1、AQZ57032.1、AQZ57103.1、A QZ57174.1, AQZ57245.1, AQZ57316.1, AQZ57387.1, AQZ57458.1, AQZ57529.1, AQZ57600.1, AQZ57671.1, AQZ57742.1, AQZ57813.1, AQZ57884.1, AQZ57 955.1, 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、AQZ5 9162.1、ARO38000.1、ARO38001.1、ARO38002.1、ARO38003.1、ARO38004.1、A RO38005.1, ARO38006.1, ARO38007.1, ARO38008.1, ARO38009.1, ARO38010.1, ARO38011.1, ARO38012.1, ARO38013.1, ARO38014.1, ARO38015.1, ARO3 8016.1, 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, A RO38039.1, 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, the inclusion of one of these residues elicits antibodies that inhibit binding to Nectin-1.

[0099] The designations used herein for gD amino acid residues include the residues of the signal sequence, thus the residue of the mature protein is designated "26."

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

[0101] In another embodiment, the HSV gD, gC, and gE proteins and fragments thereof encoded by the modified mRNA disclosed herein are described in U.S. Patent Publication No. 2013-0028925-A1, which is 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, sequence-conservative variants, and function-conservative variants of gD 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 sequence variants of HSV as found in infected individuals. 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 HSV-1 gC comprises: [ka] [ka] [ka]

[0105] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent 5' untranslated sequences. In one embodiment, the bolded residues represent a signal sequence (leader sequence) that aids in expression of the gC1 fragment. In one embodiment, the italicized residues represent 3' untranslated sequences 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 invention is the HSV-1 gC fragment set forth in the amino acid sequence Contains amino acids 27 to 457 of gC from the KOS strain: ETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGE VTPATQGMYYLAWGRMDSPHEYGTWVRVRMFRPPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHH (sequence number 8).

[0108] In one embodiment, the fragment of gC encoded by the modified mRNA used in the methods and compositions of the 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 invention comprises the following amino acid sequence: MAPGRVGLAVVLWGLLWLGAGVAGGSETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTG PLPTQRLIIGEVTPATQGMYYLAWGRMDSPHEYGTWVRVRMFRPPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHHGSHQPPPRDPTERQVIEAIEWVGIGIGVLAAGVLVVTAIVYVVRTSQSRQRHRR (sequence number 9).

[0110] In another embodiment, the gC of HSV-1 encoded by the modified mRNA used in the methods and compositions of the invention comprises an amino acid sequence set forth in any of the following GenBank Accession Nos.: 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, AD M23583.1, ADM23658.1, ADM23733.1, ADM23809.1, AEQ77075.1, AEQ77099.1, AER37628.1, AER37697.1, AER37767.1, AER37838.1, AER37910.1, AER379 81.1, 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, AF A36200.1, AFA36201.1, AFA36202.1, AFA36203.1, AFE62872.1, AFH78104.1, AFI23635.1, AFK50391.1, AFP86408.1, AGZ01906.1, AIR95840.1, AJE59 989.1, AJE60060.1, AJE60131.1, AJE60202.1, AKE48623.1, AKE98415.1, AKE98416.1, AKE98417.1, AKE98418.1, AKE98419.1, AKE98420.1, AKE98421.<h2 style=";text-align:left;direction:ltr">1、AKE98422.1、AKE98423.1、AKE98424.1、AKE98425.1、AKE98426.1、AKE98 427.1、AKE98428.1、AKE98429.1、AKE98430.1、AKE98431.1、AKE98432.1、AK E98433.1, AKE98434.1, AKE98435.1, AKG59227.1, AKG59299.1, AKG59372.1, AKG59444.1, AKG59516.1, AKG59591.1, AKG59663.1, AKG59736.1, AKG598 07.1, 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、AKG610 32.1、AKG61104.1、AKG61175.1、AKG61248.1、AKG61321.1、AKG61392.1、AKG 61464.1、AKG61537.1、AKG61611.1、AKG61684.1、AKG61756.1、AKG61828.1 、AKG61902.1、AKG61974.1、AKH80444.1、AKH80517.1、AKM76368.1、ALM2261 3.1, 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、ANN8447 8.1、ANN84555.1、ANN84632.1、ANN84708.1、ANN84785.1、ANN84861.1、ANN8 4938.1、ANN85014.1、ANN85091.1、ANN85167.1、ANN85242.1、ANN85319.1、A NN85396.1、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, B AM73394.1, CAA32294.1, CAB40083.1, CAD13356.1, CAD13357.1, CAD13358.1, CAD13359.1, CAD13360.1, CA D13361.1, CAD13362.1, CAD13363.1, CAD13364.1, CAD13365.1, CAD13366.1, CAD13367.1, CAD13368.1, CAD1 3369.1, CAD13370.1, CAD13371.1, CAD13372.1, CAD13373.1, CAD13374.1, CAD13375.1, CAD13376.1, CAD13 377.1, CAD13378.1, P04290.1, P04488.1, P09855.1, P10228.1, P28986.1, SBO07729.1, SBO07793.1, SBO077 98.1, 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: [ka] [ka] [ka]

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

[0114] In another embodiment, the nucleotide sequence of the modified mRNA encoding a 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 present invention is the HSV-2 gC fragment set forth in the amino acid sequence Amino acids 27 to 426 of gC from strain 333 include: 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: MALGRVGLAVGLWGLLWVGVVVLANASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLET QGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHHGSHQPPPRDPTERQVIRAVEGAGIGVAVLVAVVLAGTAVVYLTHASSVRYRRLR (sequence number 12).

[0117] In another embodiment, the gC of HSV-2 encoded by the modified mRNA used in the methods and compositions of the invention comprises an amino acid sequence set forth in any of the following GenBank Accession Nos.: 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, AM B66151.1, AMB66224.1, AMB66252.1, AMB66253.1, AMB66368.1, AMB66441. 1, AQZ55735.2, AQZ55806.1, AQZ55877.1, AQZ55948.1, AQZ56019.1, AQZ560 90.1, AQZ56161.2, AQZ56232.2, AQZ56303.2, AQZ56374.2, AQZ56445.1, AQ Z56516.1, AQZ56587.1, AQZ56658.1, AQZ56729.2, AQZ56800.1, AQZ56871. 1, AQZ56942.2, AQZ57013.1, AQZ57084.2, AQZ57155.1, AQZ57226.1, AQZ57 297.1, AQZ57368.1, AQZ57439.1, AQZ57510.1, AQZ57581.1, AQZ57652.1, AQ Z57723.1, AQZ57794.2, AQZ57865.2, AQZ57936.1, AQZ58007.2, AQZ58078. 1, AQZ58149.2, AQZ58220.1, AQZ58291.1, AQZ58362.1, AQZ58433.1, AQZ58 504.1, AQZ58575.1, AQZ58646.1, AQZ58717.2, AQZ58788.2, AQZ58859.2, A QZ58930.1, 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, CA B96544.1, 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 contains a properdin-interfering domain, which in one embodiment refers to a domain that blocks or inhibits host C3b molecules from binding to host properdin molecules. In another embodiment, the 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 interference domain. In another embodiment, the fragment of the gC protein is a portion of the C5 interference domain. In another embodiment, the "C5 interference domain" refers to a domain that interferes with the binding of a host C3b molecule to a host C5 molecule. In another embodiment, the 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 a separate embodiment of the present invention.

[0121] In another embodiment, the fragment of 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-conservative variants, and function-conservative 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 include an immunoprotective gC antigen from any strain of HSV. In another embodiment, the immunogenic fragment can include sequence variants of HSV as found in infected individuals. 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 HSV-1 gD comprises: [ka] [ka] [ka]

[0124] In one embodiment, all uridine residues are 1-methyl-pseudouridine. In one embodiment, the underlined residues represent 5' untranslated sequences. In one embodiment, the bolded residues represent a signal sequence (leader sequence) that aids in expression of the gEl fragment. In one embodiment, the italicized residues represent 3' untranslated sequences 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 present invention is the HSV-1 gE fragment set forth in the amino acid sequence Amino acids 24 to 409 of gE from the NS strain include: 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 to 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: (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 invention comprises an amino acid sequence set forth in any of the following GenBank Accession Nos.: 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, AD M23833.1, ADN34689.1, ADN34692.1, ADN34695.1, AEQ77099.1, AER37649.1, AER37717.1, AER37788.1, AER37859.1, AER37931.1, AER38002.1, AER380 72.1, 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, AF A36201.1, AFA36202.1, AFA36203.1, AFE62896.1, AFI23659.1, AFK50417.1, AFP86432.1, AGZ01930.1, AIR95859.1, AJE60011.1, AJE60082.1, AJE60 153.1, AJE60224.1, AJE60295.1, AKE48647.1, AKE98373.1, AKE98374.1, AKE98375.1, AKE98376.1, AKE98377.1, AKE98378.1, AKE98379.1, AKE98380.<h2 style=";text-align:left;direction:ltr">1、AKE98381.1、AKE98382.1、AKE98383.1、AKE98384.1、AKE98385.1、AKE98 386.1、AKE98387.1、AKE98388.1、AKE98389.1、AKE98390.1、AKE98391.1、AK E98392.1, AKE98393.1, AKG59248.1, AKG59320.1, AKG59393.1, AKG59464.1, AKG59538.1, AKG59611.1, AKG59684.1, AKG59757.1, AKG59828.1, AKG599 00.1、AKG59974.1、AKG60048.1、AKG60120.1、AKG60191.1、AKG60263.1、AK G60336.1、AKG60406.1、AKG60476.1、AKG60548.1、AKG60622.1、AKG60694.1 、AKG60765.1、AKG60837.1、AKG60908.1、AKG60980.1、AKG61052.1、AKG611 25.1、AKG61196.1、AKG61269.1、AKG61341.1、AKG61413.1、AKG61486.1、AKG 61558.1, AKG61631.1, AKG61705.1, AKG61776.1, AKG61849.1, AKG61922.1, AKG61995.1, AKH80465.1, AKH80538.1, ALM22637.1, ALM22711.1, ALM2278 5.1, 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、ANN8450 2.1、ANN84579.1、ANN84655.1、ANN84732.1、ANN84808.1、ANN84885.1、ANN8 4961.1、ANN85038.1、ANN85114.1、ANN85189.1、ANN85266.1、ANN85343.1、A NN85418.1、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, C AF24761.1, CAF24762.1, CAF24763.1, CAF24764.1, CAF24765.1, CAF24766.1, CAF24767.1, CAF24768.1, CAF 24769.1, CAF24770.1, CAF24771.1, CAF24772.1, CAF24773.1, CAF24774.1, CAF24775.1, CAF24776.1, CAF2 4777.1, CAF24778.1, CAF24779.1, CAF24780.1, CAF24781.1, CAF24782.1, CAF24783.1, CAF24784.1, CAF247 85.1, 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 HSV-2 gE comprises: [ka] [ka] [ka]

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

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

[0134] In one embodiment, a fragment of HSV-2 gE encoded by a modified mRNA used in the methods and compositions of the invention includes amino acids 24-405 of gE from HSV-2 strain 2.12, as set forth in the following amino acid sequence: RTSWKRVTSGEDVVLLPAPAGPEERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAP VPTPTPDDYDEEDDAGVSERTPVSVPPPTPPRRPPVAPPTHPRVIPEVSHVRGVTVHMETPEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCAEMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRA (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 invention comprises the following amino acid sequence: (SEQ ID NO:18).

[0136] In another embodiment, the HSV-2 gE encoded by the modified mRNA used in the methods and compositions of the invention comprises an amino acid sequence set forth in any of the following GenBank Accession Nos.: 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, ABW 83348.1, ABW83350.1, ABW83352.1, ABW83354.1, ABW83356.1, ABW83358.1, ABW83360.1, ABW83362.1, ABW83364.1, ABW83366.1, ABW83368.1, ABW8337 0.1, 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, AKC 59520.1, AKC59591.1, AMB66104.1, AMB66173.1, AMB66246.1, AMB66465.1 , AQZ55756.1, , AQZ55827.1, AQZ55898.1, AQZ55969.2, AQZ56040.2, AQZ56 111.2, AQZ56182.1, AQZ56253.2, AQZ56324.1, AQZ56395.1, AQZ56466.2, A QZ56537.1, AQZ56608.1, AQZ56679.1, AQZ56750.1, AQZ56821.2, AQZ56892.1, AQZ56963.2, AQZ57034.2, AQZ57105.1, AQZ57176.1, AQZ57247.2, A QZ57318.2, AQZ57389.2, AQZ57460.2, AQZ57531.2, AQZ57602.2, AQZ57 673.1, 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, AQZ 58809.2, AQZ58880.2, AQZ58951.2, AQZ59022.2, AQZ59093.1, AQZ5916 4.1, 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 gE fragment encoded by the modified mRNA used in the methods and compositions of the invention comprises the IgG Fc binding domain of the gE protein, hi another embodiment, the gE domain encoded by the modified mRNA used in the methods and compositions of the invention is any other gE domain known in the art that mediates binding to the Fc of IgG.

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

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

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

[0141] In another embodiment, the fragment of gE protein encoded by the modified mRNA used in the methods and compositions of the 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-conservative variants, and function-conservative variants of gE 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 gE antigen from any strain of HSV. In another embodiment, the immunogenic fragment can include sequence variants of HSV as found in infected individuals.

[0142] In one embodiment, the HSV glycoproteins encoded by the modified mRNAs used in the methods and compositions of the invention are homologs of the sequences provided herein. In another embodiment, the HSV glycoproteins encoded by the modified mRNAs used in the methods and compositions of the invention are isoforms of the sequences provided herein. In another embodiment, the HSV glycoproteins encoded by the modified mRNAs used in the methods and compositions of the invention are variants of the sequences provided herein. In another embodiment, the HSV glycoproteins encoded by the modified mRNAs used in the methods and compositions of the invention are fragments of the sequences provided herein.

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

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

[0145] In one embodiment, "variant" refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue) that differs from the majority of the population but is sufficiently similar to the common form to still be considered one of them, e.g., a splice variant. In one embodiment, a variant may be a sequence-conservative variant, while in another embodiment, a variant may be a function-conservative variant. In one embodiment, a variant may include an addition, deletion, or substitution of one or more amino acids.

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

[0147] The present invention also provides modified mRNAs that encode analogs of HSV proteins or polypeptides, or fragments thereof. Analogs can differ from the naturally occurring proteins or peptides by conservative amino acid sequence substitutions, or by modifications that do not affect sequence, or by both.

[0148] In another embodiment, the HSV glycoproteins encoded by the modified mRNAs of the invention are homologous to the sequences set forth above, either explicitly or by reference to GenBank entries. The terms "homologous," "homologs," and the like, when referring to any protein or peptide, in one embodiment, refer to the percentage of amino acid residues in a candidate sequence that match those of the corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and any conservative substitutions are not considered part of the sequence identity. Alignment methods and computer programs are well known in the art.

[0149] In another embodiment, "homology" refers to greater than 70% identity of the protein sequence encoded by the modified mRNA 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" refers to a variant of a molecule, e.g., a protein, that differs only slightly from another isoform of the same protein. In one embodiment, isoforms can be produced from different but related genes, or in another embodiment, can arise from the same gene by alternative splicing. In another embodiment, isoforms arise due to single nucleotide polymorphisms.

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

[0152] In another embodiment, the methods and compositions of the invention utilize chimeric molecules, which include a fusion of a modified mRNA encoding an HSV protein with a modified mRNA encoding a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. In another embodiment, the epitope tag is located at the amino-terminus or carboxy-terminus of the protein, or at an internal location therein. The presence of such epitope-tagged forms of recombinant HSV proteins is detected, in another embodiment, by an antibody against the tag polypeptide. In another embodiment, the inclusion of the epitope tag allows for the recombinant HSV protein to be readily purified 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 invention includes an adjuvant, while in another embodiment, the composition does not include an adjuvant. "Adjuvant" refers, in another embodiment, to a compound that, when administered to an individual or tested in vitro, increases the immune response to an antigen in an individual or test system to which the antigen is administered. In another embodiment, an immune adjuvant promotes an immune response to an antigen that is weakly immunogenic when administered alone, i.e., elicits no or weak antibody titers or elicits a cell-mediated immune response. In another embodiment, an adjuvant increases the antibody titer to an antigen. In another embodiment, an adjuvant reduces the dose of an antigen that is effective to achieve an immune response in an individual. Several types of adjuvants are known in the art and are described in detail in U.S. Patent Publication No. 2013 / 0028925, which is incorporated herein by reference. modified mRNA

[0154] In one embodiment, the invention provides compositions comprising and methods of use of modified mRNA, hi one embodiment, the modified mRNA comprises one or more modified nucleoside residues.

[0155] In another embodiment, the modified nucleoside of methods and compositions 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 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-glycinylcarbamoyl adenosine), t 6 A (N6-threonylcarbamoyl adenosine), ms 2 t 6 A(2-methylthio-N6-threonylcarbamoyl adenosine), m 6 t 6 A (N6-methyl-N6-threonylcarbamoyl adenosine), hn 6 A (N6-hydroxynorvalylcarbamoyl adenosine), ms 2 hn 6 A (2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine), 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 hydroxy-wybutosine), imG (wybutosine), mimG (methylwybutosine), Q (queosine), oQ (epoxyqueosine), galQ (galactosyl-queosine), manQ (mannosyl-queosine), 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 U (5-cyanomethyl-uridine).

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

[0158] In one embodiment, the modified mRNA comprises a pseudouridine residue. 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 residues include 1-methylpseudouridine residues 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-methylpseudouridine), 1-methyl- ... 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 3Examples of pseudouridine include, but are not limited to, 2'-O-methyl-pseudouridine (Ψm).

[0160] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having 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 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-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 modified cytosines include 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, lysine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-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 modified adenines include 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 a modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OHyW), modified hydroxywyosine (OHyW * ), 7-deaza-guanosine, queosine (Q), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (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-ribosylguanosine(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, nucleobases may also be used, for example, pyrazole[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-azouracil, 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 and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazole[3,4-d]pyrimidines, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazines, pyridazines; and 1,3,5 triazines. When nucleotides are represented using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives, e.g., A includes adenine or an adenine analog such as 7-deazaadenine. Modifications in internucleoside linkages

[0165] Modified nucleotides may be incorporated into polynucleotides, primary constructs, or mRNA molecules and may be modified in the internucleoside linkage (e.g., the phosphate backbone). The phrases "phosphate" and "phosphodiester" are used interchangeably herein in the context of polynucleotide backbones. The backbone phosphate group may be modified by replacing one or more of the oxygen atoms with different substituents. Modified nucleosides and nucleotides may also include extensive replacement of the unmodified phosphate moiety with alternative internucleoside linkages as described herein. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, and phosphate esters. phosphonate), phosphoramidate, phosphorodiamidate, alkyl or aryl These include, but are not limited to, phosphonates and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced with sulfur. Phosphate linkers can also be modified by replacing the linking oxygens with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0166] The α-thio substituted phosphate moieties are provided to provide stability to RNA and DNA polymers through unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently longer half-life in cellular environments. Phosphorothioate linked polynucleotides, primary constructs, or mmRNA molecules are also expected to reduce innate immune responses due to weaker binding / activation of cellular 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 (α-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 according to the present invention, including internucleoside linkages that do not contain a phosphorus atom, are described herein below. Combinations of modified sugars, nucleobases, and internucleoside linkages

[0169] The polynucleotides, primary constructs, and mmRNA of the invention can comprise a combination of modifications to the sugar, nucleobase, and / or internucleoside linkage.

[0170] In another embodiment, a purified preparation of RNA, oligoribonucleotides, or polyribonucleotides of methods and compositions of the invention comprises a combination of two or more of the modifications described above. In another embodiment, a purified preparation of RNA or oligoribonucleotides comprises a combination of three or more of the modifications described above. In another embodiment, a purified preparation of RNA or oligoribonucleotides comprises a combination of more than three of the modifications described above.

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

[0172] In one embodiment, the invention comprises one or more modified mRNAs encoding an HSV glycoprotein. In one embodiment, the modified RNA comprises a pseudouridine or pseudouridine family residue. In another embodiment, the modified mRNA of the invention is capable of directing the protein expression of the HSV glycoprotein encoded therein.

[0173] In another embodiment, the invention provides in vitro transcribed mRNA molecules encoding HSV glycoproteins that contain pseudouridine.In another embodiment, the invention provides synthetic mRNA molecules encoding HSV glycoproteins that contain pseudouridine.

[0174] In another embodiment, the in vitro transcribed mRNA molecules of the methods and compositions of the invention are synthesized by T7 phage RNA polymerase. In another embodiment, the molecules are synthesized by SP6 phage RNA polymerase. In another embodiment, the molecules are synthesized by T3 phage RNA polymerase. In another embodiment, the molecules are synthesized by a polymerase selected from those polymerases listed above. In another embodiment, the mRNA is chemically synthesized on a cylinder similar to DNA.

[0175] In another embodiment, the modified nucleoside in the RNA, oligoribonucleotide, or polyribonucleotide of methods and compositions of the present 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 methods and compositions of the present invention further comprises a poly-A tail. In another embodiment, the modified mRNA of methods and compositions of the present invention does not comprise a poly-A tail. Each possibility represents a separate embodiment of the present invention.

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

[0178] In another embodiment, the modified mRNA of methods and compositions of the present invention further comprises a cap-independent translation enhancer. In another embodiment, the modified mRNA of 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 tobacco etch virus (TEV) cap-independent translation enhancer. In another embodiment, the cap-independent translation enhancer is any other cap-independent translation enhancer known in the art. Each possibility represents a separate embodiment of the present invention.

[0179] In one embodiment, "pseudouridine" is 1 acp 3 In another embodiment, the term refers to m 1 In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 In another embodiment, the term refers to m 3Ψ (3-methylpseudouridine). In another embodiment, the modified nucleoside is 4' (pseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the pseudouridines listed above. In another embodiment, the term refers to other pseudouridines known in the art. Each possibility represents a separate embodiment of the present invention.

[0180] In another embodiment, the modified RNA comprises modified nucleosides, which in one embodiment are 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 of delivering a recombinant protein to a subject, comprising contacting a subject with a 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 invention comprises increasing the number, proportion or frequency of modified uridine nucleosides in an RNA molecule to reduce immunogenicity or increase the efficiency of translation, hi one embodiment, the number of modified uridine residues in an RNA, oligoribonucleotide or polyribonucleotide molecule determines the magnitude of the effect observed in the present invention.

[0183] In another embodiment, between 0.1% and 100% of the uridine residues in the modified mRNA of 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, 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0184] 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 8%. In another embodiment, the percentage is less than 10%. 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%.

[0185] In another embodiment, 0.1% of the residues of a given uridine nucleotide are modified. In another embodiment, the percentage of nucleotides is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage 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 polyribonucleotide refer to compounds that contain nucleotides in which, in one embodiment, the sugar moiety is ribose. In another embodiment, the term includes both RNA and RNA derivatives with modified backbones. A variety of RNA backbone modifications are known in the art and are contemplated in the present invention. In one embodiment, the modified RNA is a PNA (peptide nucleic acid). PNAs containing peptide backbones and nucleotide bases can bind to both DNA and RNA molecules in another embodiment. In another embodiment, the nucleotides are modified by replacement of one or more phosphodiester bonds with phosphorothioate bonds. In another embodiment, the artificial nucleic acids contain other variants of the phosphate backbones of natural nucleic acids known in the art. Each nucleic acid derivative represents a separate embodiment 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. Patent Nos. 5,723,335 and 5,663,153 issued to Hutcherson et al., and related PCT publication WO 95 / 26204, each of which represents a separate embodiment of the present invention.

[0189] The nucleic acid of interest can be purified by any method known in the art or to be developed that removes contaminants from a preparation of the nucleic acid, thereby substantially reducing the immunogenic potential 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 liquid chromatographic separation and enzymatic digestion, either alone or in any combination, simultaneously or in any order. Non-limiting examples of liquid chromatographic separation include HPLC and fast protein liquid chromatography (FPLC). Materials useful in the HPLC and FPLC methods of the present invention include cross-linked polystyrene / divinylbenzene (PS / DVB), PS / DVB-C18, PS / DVB-alkylated, Helix DNA columns (Varian), Eclipse dsDNA Analysis columns (Agilent Technologies). Enzymes useful in the enzymatic digestion methods of the invention include any enzyme capable of digesting any contaminants, e.g., dsRNA contaminants, in the nucleic acid formulations of the invention, and include RNase III, RNase VI, Dicer, and Chipper (see Fruscoloni et al., 2002, PNAS 100:1639). Non-limiting examples of assays to assess the purity of a nucleic acid of interest include dot blot assays, Northern blot assays, and dendritic cell activation assays, as described elsewhere herein.

[0190] In another embodiment, the modified mRNA of the methods and compositions of the invention is significantly less immunogenic than an unmodified in vitro synthesized mRNA molecule of the same sequence. In another embodiment, the modified mRNA molecule is 2-fold less immunogenic than its unmodified counterpart. In another embodiment, immunogenicity is reduced 3-fold. In another embodiment, immunogenicity is reduced 5-fold. In another embodiment, immunogenicity is reduced 7-fold. In another embodiment, immunogenicity is reduced 10-fold. In another embodiment, immunogenicity is reduced 15-fold. In another embodiment, immunogenicity is reduced by a factor. In another embodiment, immunogenicity is reduced 50-fold. In another embodiment, immunogenicity is reduced 100-fold. In another embodiment, immunogenicity is reduced 200-fold. In another embodiment, immunogenicity is reduced 500-fold. In another embodiment, immunogenicity is reduced 1000-fold. In another embodiment, immunogenicity is reduced 2000-fold. In another embodiment, immunogenicity is reduced by another factor difference.

[0191] In another embodiment, "significantly less immunogenic" refers to a detectable reduction in immunogenicity. In another embodiment, the term refers to a fold reduction in immunogenicity (e.g., one of the fold reductions listed above). In another embodiment, the term refers to a reduction such that an effective amount of the modified mRNA can be administered without eliciting a detectable immune response. In another embodiment, the term refers to a reduction such that the modified mRNA can be administered repeatedly without eliciting an immune response sufficient to detectably reduce expression of the recombinant protein. In another embodiment, the reduction is such that the modified mRNA can be administered repeatedly without eliciting an immune response sufficient to elicit detectable expression of the recombinant protein.

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

[0193] In another embodiment, the modified mRNA of the methods and compositions of the invention is translated in a cell more efficiently than an unmodified mRNA molecule of the same sequence. In another embodiment, the modified mRNA exhibits enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced 2-fold compared to its unmodified counterpart. In another embodiment, translation is enhanced 3-fold. In another embodiment, translation is enhanced 5-fold. In another embodiment, translation is enhanced 7-fold. In another embodiment, translation is enhanced 10-fold. In another embodiment, translation is enhanced 15-fold. In another embodiment, translation is enhanced 20-fold. In another embodiment, translation is enhanced 50-fold. In another embodiment, translation is enhanced 100-fold. In another embodiment, translation is enhanced 200-fold. In another embodiment, translation is enhanced 500-fold. In another embodiment, translation is enhanced 1000-fold. In another embodiment, translation is enhanced 2000-fold. In another embodiment, the ratio is between 10-1000-fold. In another embodiment, the rate is 10-100 fold. In another embodiment, the rate is 10-200 fold. In another embodiment, the rate is 10-300 fold. In another embodiment, the rate is 10-500 fold. In another embodiment, the rate is 20-1000 fold. In another embodiment, the rate is 30-1000 fold. In another embodiment, the rate is 50-1000 fold. In another embodiment, the rate is 100-1000 fold. In another embodiment, the rate is 200-1000 fold. In another embodiment, translation is promoted by other significant amounts or ranges of amounts. Each possibility represents a separate embodiment of the present invention.

[0194] Methods for determining translation efficiency are well known in the art and can include, for example, measuring the activity of an encoded reporter protein (e.g., luciferase or Renilla or green fluorescent protein [Wall AA, Phillips AM et al, Effective translation of the second cistron in two Drosophila dicistronic transcripts is determined by the absence of in-frame AUG codons in the first cistron. J Biol Chem 2005; 280(30): 27670-8]), or measuring radiolabel incorporated into the translated protein (Ngosuwan J, Wang NM et al, Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of pre-secretory proteins into the endoplasmic reticulum. J Biol Chem 2003; 278(9): 7034-42). Each method represents a separate embodiment of the present invention.

[0195] In another embodiment, the labeled cell of the methods of the invention is a dendritic cell. In another embodiment, the labeled cell of the methods of the invention is a macrophage. In another embodiment, the labeled cell of the methods of the invention is a B cell. In another embodiment, the labeled cell of the methods of the invention is another antigen presenting cell. In another embodiment, the labeled cell of the methods of the invention is a mucosal cell. In another embodiment, the labeled cell of the methods of the invention is an epithelial cell. In another embodiment, the cell is a skin cell. In another embodiment, the cell is an epithelial cell. In another embodiment, the cell is a keratinocyte. In another embodiment, the cell is a Merkel cell, a melanocyte, or a Langerhans cell. Each method represents a separate embodiment of the present invention.

[0196] Methods of Treatment and Use of the Compositions The invention also provides a method of vaccinating a subject against HSV and treating, preventing, inhibiting, reducing the occurrence of, or suppressing an HSV infection or a symptom or sign thereof, comprising administering a composition of the invention.

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

[0198] 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 which encodes an HSV glycoprotein or an immunogenic fragment thereof.

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

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

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

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

[0203] In accordance with this aspect, in one embodiment, the invention provides a method of treating, suppressing, inhibiting or reducing the incidence of herpes simplex virus 1 (HSV-1) infection in a subject, comprising contacting the subject with a composition comprising one or more modified mRNAs, each of which encodes 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 incidence of herpes simplex virus 2 (HSV-2) infection in a subject, comprising contacting the subject with a composition comprising one or more modified mRNAs, each of which encodes an HSV-2 glycoprotein or an immunogenic fragment thereof.

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

[0206] In another embodiment, the present invention provides a method of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject, comprising administering to the subject an immunogenic composition comprising a modified mRNA encoding: (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 combination thereof.

[0207] In another embodiment, the present invention provides a method of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject, comprising administering to the subject an immunogenic composition comprising a modified mRNA encoding: (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 combination thereof.

[0208] In another embodiment, the present invention provides a method of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject, comprising administering to the subject an immunogenic composition comprising a modified mRNA encoding: (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 combination thereof.

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

[0210] In another embodiment, the present invention provides a method of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject, comprising administering to the subject an immunogenic composition comprising a modified mRNA encoding: (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 combination thereof.

[0211] In another embodiment, the present invention provides a method of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject, comprising administering to the subject an immunogenic composition comprising a modified mRNA encoding: (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 combination thereof.

[0212] In another embodiment, the invention provides a method of inhibiting initial HSV infection in a subject comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of treating HSV infection in a subject comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of reducing the incidence of HSV infection in a subject comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of inhibiting initial HSV infection in a subject comprising administering to the subject a composition of the invention. Relapse The present invention provides a method of inhibiting a tumor in a subject, the method comprising administering to the subject a composition of the present invention.

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

[0214] In one embodiment, " Relapse " or "recurrence" refers to reinfection of skin tissues following latent HSV infection of neural cells. In another embodiment, the term refers to reactivation of HSV after a latent period. In another embodiment, the term refers to symptomatic HSV lesions after a non-symptomatic latent period.

[0215] In another embodiment, the present invention provides a method for 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, the anterograde spread is inhibited. In one embodiment, the retrograde spread is inhibited. "DRG" ​​refers to the neuronal cell body in one embodiment, and contains the neuronal cell body of the nerve fiber 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 invention provides a method of inhibiting recurrence after an initial HSV infection in a subject, comprising administering to the subject a composition of the invention.In another embodiment, the invention provides a method of preventing recurrence after an initial HSV infection in a subject, comprising administering to the subject a composition of the invention.

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

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

[0219] In another embodiment, the present invention provides a method of treating HSV encephalitis in a subject, comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method of reducing the incidence of HSV encephalitis in a subject, comprising administering to the subject a composition of the present invention. "HSV encephalitis" refers, in one embodiment, to encephalitis caused by Herpes Simplex Virus-1 (HSV). 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 of treating or reducing HSV neonatal infection in a subject, comprising administering to said subject a composition of the present invention.

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

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

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

[0224] "HSV-1" refers, in another embodiment, to Herpes Simplex Virus-1. 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, in another embodiment, to herpes simplex virus-2. In another embodiment, the term refers to HSV-2 strain 333. In another embodiment, the term refers to strain 2.12. In another embodiment, the term refers to strain HG52. In another embodiment, the term refers to strain MS. In another embodiment, the term refers to strain G. In another embodiment, the term refers to strain 186. In another embodiment, the term refers to any other HSV-2 strain known in the art.

[0226] In another embodiment, the invention provides a method of vaccinating a subject against HSV infection comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of inhibiting HSV infection in a subject comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of preventing HSV infection in a subject comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of preventing initial HSV infection in a subject comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of preventing neuronal HSV transmission in a subject comprising administering to the subject a composition of the invention.

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

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

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

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

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

[0233] In another embodiment, the herpes encephalitis treated or prevented by the methods of the present invention is focal 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 of treating or reducing the occurrence of a disease, disorder, or condition associated with or secondarily associated with HSV encephalitis in a subject, comprising administering to the subject a composition of the present invention.

[0235] In another embodiment, the invention provides a method of treating an HSV infection, reducing the pathogenicity of an HSV infection, ameliorating symptoms of an HSV infection, ameliorating secondary symptoms of an HSV infection, reducing the incidence of an HSV infection, or prolonging the latency to recurrence of an HSV infection in a subject, comprising administering to the subject a composition of the invention.

[0236] In another embodiment, the invention provides a method of protecting a subject against the formation of shingles lesions or similar outbreaks in a human subject.In another embodiment, the invention provides a method of inhibiting the formation of HSV shingles lesions or similar outbreaks in a human subject.

[0237] "Zoster" in one embodiment refers to the skin lesions characteristic of HSV infection, particularly during reactivation infection, which in one embodiment begin as a rash followed by distribution around the dermatomes, generally occurring in a stripe or belt-like pattern. In one embodiment, the rash develops into vesicles or small blisters filled with serous fluid. In one embodiment, shingles lesions form in mice as a result of contact with HSV. In another embodiment, shingles lesions form in humans as a result of contact with HSV. "Zoster transmission" in one embodiment refers to HSV infection that spreads from a ganglion into secondary skin sites within the dermatome. In another embodiment, the term refers to transmission within the same dermatome as the initial site of infection. In another embodiment, the term refers to any other definition of "Zoster transmission" known in the art. "Outbreak" refers, in another embodiment, to a sudden increase in symptoms of a disease or in the transmission or spread of a disease, and in one embodiment to a sudden increase in shingles lesions, while in another embodiment, "outbreak" refers to a sudden eruption of shingles lesions.

[0238] In one embodiment, the present invention provides a method for preventing the formation of dermatomal lesions or similar conditions in a subject. In one embodiment, dermatomal lesions form as a result of contact with HSV. In another embodiment, dermatomal lesions most frequently occur when the virus reactivates from latency in a ganglion, and in one embodiment, spreads down the nerves, causing recurrent infections in one embodiment.

[0239] It should be understood that the method of the present invention can be used to treat, inhibit, suppress, etc., HSV infection or primary or secondary symptoms associated with such infection after a subject is exposed to HSV. In another embodiment, the subject is infected with HSV before vaccination. In another embodiment, the subject is at risk for HSV infection. In another embodiment, vaccination with the method 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 therapeutic treatment or preventive or prophylactic treatment, the purpose being to prevent or alleviate the targeted pathological condition or disorder described herein above.Thus, in one embodiment, treating can directly affect or cure, suppress, inhibit, prevent, reduce severity, delay onset, reduce symptoms associated with disease, disorder or condition, or combinations thereof.Thus, in one embodiment, "treating" refers to, among other things, delaying progression, promoting remission, inducing remission, increasing remission, accelerating recovery, increasing the effectiveness of alternative treatments, or reducing resistance to alternative treatments, or combinations thereof.In one embodiment, "preventing" refers to, among other things, delaying the onset of symptoms, preventing recurrence to disease, reducing the number or frequency of recurrent episodes, increasing the latency between symptomatic episodes, or combinations thereof. In one embodiment, "suppressing" or "inhibiting" refers to, among other things, reducing the severity of a symptom, reducing the severity of an acute episode, reducing the number of symptoms, reducing the occurrence of symptoms associated with a disease, reducing the latency of a symptom, ameliorating a symptom, 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 in reducing 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 in treating or inhibiting genital ulcer disease, which in one embodiment entails reducing the severity or frequency of HSV genital ulcer 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 may generate high titers of neutralizing antibodies or strong T cell responses, but upon 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 enhancing the effectiveness of gD-1 subunit compositions, in one embodiment, using gC-1 to prevent immune evasion from complement.

[0242] In one embodiment, studies in guinea pigs and mice suggest that viral load in ganglion correlates with the frequency of recurrent HSV infection.Thus, in one embodiment, the compositions and methods of the present invention are useful for preventing or inhibiting recurrent HSV infection.In one embodiment, for example, antibodies against gC-1 block domains involved in immune evasion, enhance complement activity, improve the neutralizing activity of anti-gD-1, increase antibody and complement-dependent cytotoxicity activity, and increase complement-mediated neutralization and lysis of infected cells.

[0243] In one embodiment, the symptoms are primary, while in another embodiment, the symptoms are secondary.In one embodiment, "primary" refers to symptoms that are the direct result of viral infection of a subject, while in one embodiment, "secondary" refers to symptoms that are brought about or are the result of a 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 an HSV infection, including blisters, ulcers, or lesions on the urethra, cervix, upper thighs, and / or anus in women or on the penis, urethra, scrotum, upper thighs, and anus in men, inflammation of the tongue, mouth or lips, swelling, fever, flu-like symptoms, canker sores, sore throat, pharyngitis, pain, blisters, ulcers, cold sores, neck pain, enlarged lymph nodes, redness, bleeding, itching, difficulty urinating, headache, muscle pain, and the like, or a combination 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 stiff neck. In another embodiment, the disease, disorder, or symptom is seizures. In another embodiment, the disease, disorder, or symptom is hemiplegia. In another embodiment, the disease, disorder, or symptom is stupor. In another embodiment, the disease, disorder, or symptom is coma. 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 and are described, for example, in Bonkowsky JL et al., (Herpes simplex virus central nervous system relapse during treatment of infantile spasms with corticotropin, Pediatrics, May 2006;117(5):e1045-8) and Khan OA et al., (Herpes encephalitis presenting as mild aphasia: case report, BMC FamPract, Mar. 24, 2006; 7:22. Each method represents a separate embodiment of the present invention.

[0247] In another embodiment, the present invention provides a method of treating or reducing the occurrence of a disease, disorder, or symptom associated with HSV infection in a subject, 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] Thus, in one embodiment, the compositions and methods of the invention treat, suppress, inhibit or reduce the occurrence of the infection itself, while in another embodiment, the compositions and methods of the invention treat, suppress, inhibit or reduce the occurrence of the primary symptoms of the infection, while in another embodiment, the compositions and methods of the 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 invention may affect any combination of the infection, the primary symptoms caused by the infection, and the secondary symptoms associated with the infection.

[0250] In another embodiment, the HSV infection treated or ameliorated by the methods and compositions of the present invention is a genital HSV infection. 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 invention provides a method of reducing the incidence of disseminated HSV infection in a subject, comprising administering to said subject a composition of the invention.

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

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

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

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

[0256] In one embodiment, the present invention provides a method of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject infected with HIV, 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 of treating, suppressing, inhibiting, or reducing the incidence of HSV infection in a subject infected with HIV, comprising administering to the subject a composition comprising: (a) a modified mRNA encoding an HSV gC protein or a fragment thereof, the fragment comprising either a C3b-binding domain thereof, a properdin-interfering domain thereof, a C5-interfering domain thereof, or a fragment of the C3b-binding domain, a properdin-interfering domain, or a C5-interfering 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, 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, 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, comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for reducing the incidence of HSV infection in a subject infected with HIV, comprising administering to the subject a composition of the present invention. In another embodiment, the present invention provides a method for preventing HIV infection, comprising administering to the subject a HSV composition of the present invention. In one embodiment, HSV infection increases the risk of HIV infection, and protection against HSV infection reduces the risk of HIV infection. Thus, in one embodiment, the present invention provides a method for reducing the risk of HIV infection, comprising administering to the subject a composition of the present invention.

[0258] In one embodiment, the composition for use in the methods of the invention induces an immune response against HSV. In another embodiment, the composition for use in the methods of the invention induces an immune response against HSV-1. In another embodiment, the composition for use in the methods of the invention induces an immune response against HSV-2. In another embodiment, the composition comprises a modified mRNA encoding gD and gC proteins. In another embodiment, the composition comprises a modified mRNA encoding gE and gD proteins. In another embodiment, the composition comprises a modified mRNA encoding gC and gE proteins. In another embodiment, the composition comprises a modified mRNA encoding gE, gD, and gC proteins. In another embodiment, the composition comprises a 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] It should be understood that in one embodiment, the 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, may be suspected of being infected with at least one other pathogen.In one embodiment, the subject may be immunocompromised.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, the subject is a newborn, in another embodiment, immunocompromised, in another embodiment, elderly, in another embodiment, immunocompromised newborn or immunocompromised elderly subject.

[0260] In another embodiment, the compositions of the present invention and their associated uses may suppress, inhibit, prevent, or treat HIV infection in a subject. In one embodiment, the compositions of the present invention and their associated uses may treat a secondary complication of HIV infection, which in one embodiment is an opportunistic infection, a neoplasm, a neurological abnormality, or progressive immune depression. In another embodiment, the method includes treating Acquired Immune Deficiency Syndrome (AIDS). In another embodiment, the method includes treating CD4 + Treating a decrease in the number of T lymphocytes.

[0261] In another embodiment, the present invention provides a method of reducing HIV-1 transmission to offspring, comprising administering to a subject a composition of the present invention. As is known in the art, HSV-2 infection increases HIV-1 viral shedding in genital secretions (Nagot N et al., Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus, N Engl J Med., Feb. 22, 2007;356(8):790-9). Thus, the method of the present invention of inhibiting HSV-2 infection is also effective in 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 present invention provides a method for reducing HIV-1 transmission to sexual partners, comprising administering to a subject a composition of the present invention. As is known in the art, HSV-2 infection increases HIV-1 viral shedding in genital secretions. Thus, the method of the present invention for inhibiting HSV-2 infection is also effective in reducing HIV-1 transmission to sexual partners. 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 of reducing susceptibility to HIV-1, comprising administering to a subject a 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, 2006 Nov. 28;20(18):2305-13). Thus, the method of the present invention of inhibiting 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] Thus, in one embodiment, the invention provides a method of inhibiting initial HSV infection in an HIV-infected subject, comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of reducing the incidence of HSV infection in an HIV-infected subject, comprising administering to the subject a composition of the invention. In another embodiment, the invention provides a method of inhibiting initial HSV infection in an HIV-infected subject, comprising administering to the subject a composition of the invention. Relapse The present invention provides a method for inhibiting HSV infection, recurrence, or bronchitis, comprising administering to a subject a composition of the present invention. In one embodiment, administration of a composition of the present invention induces an anti-HSV immune response.

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

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

[0267] Dosing and Medication Regimen The compositions of the present invention, in another embodiment, may be administered to a subject by any method known to one of skill in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, intravaginally, intranasally, intratumorally, or topically.

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

[0269] In another embodiment of the methods and compositions of the present invention, the compositions are administered orally and are therefore formulated in a form suitable for oral administration, i.e., solid or liquid preparations. 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 in a capsule. In accordance with this embodiment, the compositions of the present invention comprise a hard gelatin capsule in addition to the active compound, the inert carrier or diluent.

[0270] In other embodiments, the pharmaceutical compositions are administered by intravenous, intraarterial, or intramuscular injection of a liquid preparation. Suitable liquid preparations include solutions, suspensions, dispersions, emulsions, oils, and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and are therefore formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intraarterially and are therefore formulated in a form suitable for intraarterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly and are therefore formulated in a form suitable for intramuscular administration.

[0271] In another embodiment, the pharmaceutical compositions are administered topically to a body surface and are therefore formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, eye drops, etc. For topical administration, the compositions or physiologically acceptable derivatives thereof are prepared and applied as a solution, suspension, or emulsion in a physiologically acceptable diluent with or without a pharmaceutical carrier.

[0272] In another embodiment, the composition is administered as a suppository, for example a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of a pellet. In another embodiment, the pellet provides a controlled release of the drug over an extended period of time.

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

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

[0276] In some embodiments, any of the HSV compositions of the invention and compositions for use in the methods of the invention comprise modified mRNAs encoding HSV proteins of the invention or combinations of modified mRNAs encoding HSV proteins, in any form or embodiment described herein. In some embodiments, any of the compositions of the invention and compositions for use in the methods of the invention comprise modified mRNAs encoding HSV proteins of the invention or combinations of modified mRNAs encoding HSV proteins, in any form or embodiment described herein. In some embodiments, the compositions of the invention consist essentially of modified mRNAs encoding HSV proteins of the invention or combinations of modified mRNAs encoding HSV proteins, in any form or embodiment described herein. In some embodiments, the term "comprising" refers to the inclusion of modified mRNAs encoding other HSV proteins, as well as the inclusion of 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 modified mRNAs encoding a particular HSV protein or fragments thereof. However, other components that are not directly involved in 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 a modified mRNA encoding a particular HSV protein or fragment, or a combination of modified mRNA encoding an HSV protein or fragment of the invention, in any form or embodiment described herein.

[0277] In another embodiment, the present invention provides a composition for treating HSV-1 or a symptom or symptom thereof comprising a modified mRNA of the present invention.

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

[0279] It should be understood that the compositions and methods of the present invention may be similarly used in non-HSV herpes viruses 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 to the gD, gE, or gC proteins of HSV-1, in one embodiment, HSV-1, or in another embodiment, HSV-2, HSV-1, gD, gE, or gC proteins. In one embodiment, such compositions may be useful for suppressing, inhibiting, preventing, or treating cancer, or in another embodiment, tumors. In one embodiment, the non-HSV herpes viruses include varicella zoster virus (VZV), Epstein-Barr virus (EBV), EBNA, cytomegalovirus (CMV), and human herpes virus-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 dose. In another embodiment, the dosage is a weekly dose. In another embodiment, the dosage is a monthly dose. In another embodiment, the dosage is a yearly dose. In another embodiment, the dosage is a series of a defined number of doses. In another embodiment, the dosage is a single dose.

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

[0283] In one embodiment, the subject is immunized with one dose of the composition. In another embodiment, the subject is immunized with one dose. In another embodiment, the subject is immunized with two doses. In another embodiment, the subject is immunized with three doses. In another embodiment, the subject is immunized with four doses. In another embodiment, the subject is immunized with five doses.

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

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

[0286] In one embodiment, the method of the invention comprises one or a single 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 booster approach. In one embodiment, the method of the invention further comprises administering to the subject one or more additional doses of the nucleoside modified mRNA composition following the first administration.

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

[0288] In another embodiment, modified mRNAs encoding gC, gD, and gE are administered simultaneously following a booster administration of modified mRNA encoding gD without modified mRNA encoding gC or gE. In another embodiment, modified mRNAs encoding gC, gD, and gE are administered simultaneously following a booster administration of modified mRNA encoding gC without modified mRNA encoding gD or gE. In another embodiment, modified mRNAs encoding gC, gD, and gE are administered simultaneously following a booster administration of modified mRNA encoding gE without modified mRNA encoding gD or gC. In another embodiment, modified mRNAs encoding gC, gD, and gE are administered simultaneously following a booster administration of modified mRNA encoding gC and gD without modified mRNA encoding gE. In another embodiment, modified mRNAs encoding gC, gD, and gE are administered simultaneously following a booster administration of modified mRNA encoding gC and gE without modified mRNA encoding gD. In another embodiment, modified mRNAs encoding gC, gD, and gE are administered simultaneously following a booster administration of modified mRNAs encoding gD and gE, without modified mRNA encoding gE. In one embodiment, the booster administration is administered at the same site and by the same mode of administration as the priming administration. In another embodiment, the booster administration is administered at a different site from the priming administration, but by the same mode of administration as the priming administration. In one embodiment, the booster administration is administered at the same site from the priming administration, but by a different mode of administration. In another embodiment, the booster administration is administered at a different site from the priming administration, but by a different mode of administration.

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

[0290] In one embodiment, the effectiveness of the compositions and methods of the invention depends on the presence of complement, while in another embodiment, the compositions and methods of the invention do not depend on the presence of complement. In one embodiment, the effectiveness of some of the compositions for use in the methods of the invention depends on the presence of complement, while others do not. In one embodiment, anti-gC antibodies depend on complement for their effectiveness against HSV.

[0291] In one embodiment, complement is an important participant in innate and adaptive immunity. In one embodiment, complement activation promotes virus neutralization by particle 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 complement C3b and blocks properdin interaction with C5 and C3b, inhibiting complement activation and complement-mediated virus neutralization. In one embodiment, the complement-interacting gC-1 domain is located within amino acids 33-133 and blocks properdin binding to C5 and C3b, and in one embodiment, the complement-interacting gC-1 domain extends from amino acids 124-366 and binds directly to C3b. In one embodiment, HSV-1 gC mutant viruses lacking the C3b binding domain are more susceptible to complement-mediated virus neutralization in vitro and are less pathogenic than wild-type (WT) virus in a mouse flank model. Thus, in one embodiment, the interaction between gC-1 and C3b enhances HSV-1 pathogenicity, and in one embodiment, blocking the gC-1 domain is effective in preventing or treating HSV-1 infection.

[0292] In one embodiment, the compositions and methods of the invention are for use in human subjects, while in another embodiment, they are for use in animal subjects. In another embodiment, the subject is a mammal. In another embodiment, the subject is any organism suspected of infection with 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 male subjects. In another embodiment, the compositions and methods of the invention are effective in female subjects. In one embodiment, the compositions and methods of the invention are effective in seronegative subjects. In another embodiment, the compositions and methods of the invention are effective in seropositive subjects.

[0293] Pharmaceutical preparations In one embodiment, the method further comprises mixing the modified mRNA with a transfection reagent prior to the contacting step. In another embodiment, the method further comprises administering the modified mRNA together with a 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, which in another embodiment, increase intracellular stability, increase uptake efficiency, and improve biological activity.

[0296] In another embodiment, liposomes are hollow spherical vesicles that contain lipids arranged in the same manner as those lipids that make up cell membranes. They have an internal aqueous space for encapsulating water-soluble compounds in another embodiment and range in size from 0.05 to several microns in diameter. In another embodiment, 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 and are described, for example, in Bose et al. 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 SR et al. (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, ψ mRNA is encapsulated in nanoparticles to improve the efficiency of delivery and expression of ψ mRNA. Nanoparticle packaging involves concentrating and encapsulating RNA into particles smaller than the pores of the nuclear membrane using chemicals including poly-L-lysine and polyethylene glycol. In one embodiment, RNA is encapsulated in four nanoparticle formulations (PEI, PLL, PAE, and CK 30 PEG 10k ) will be packaged in one type.

[0300] Lipid Nanoparticles In one embodiment, the nanoparticles used in the compositions and methods of the invention comprise lipid nanoparticles as described in Cullis, P., and Hope, M, (undated), Lipid Nanoparticle Systems for Enabling Gene Therapies. Molecular therapy., 25(7), the entire contents of which are incorporated by reference herein.

[0301] In one embodiment, the delivery of nucleoside modified RNA comprises any suitable delivery method, including the typical RNA transfection method described elsewhere herein.In some embodiments, the delivery of nucleoside modified RNA to a subject comprises mixing nucleoside modified RNA with transfection reagent prior to contacting step.In another embodiment, the method of the present invention further comprises administering nucleoside modified RNA together with 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 polyethylenimine-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] Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency, and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles containing lipids arranged in the same manner as those lipids that make up the cell membrane. They, in another embodiment, 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, 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 as described herein.For example, in one embodiment, the composition comprises LNPs and one or more nucleoside 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 order of a nanometer (e.g., 1 to 1,000 nm) that comprises one or more lipids, e.g., lipids of formula (I), (II) or (III), as described in WO2016176330A1, the entirety of which is incorporated by reference herein.

[0307] In some embodiments, lipid nanoparticles are included in the formulations that contain nucleoside modified RNA as described herein.In some embodiments, such lipid nanoparticles contain one or more excipients selected from cationic lipid and neutral lipid, charged lipid, steroid and polymer-bound lipid (e.g., PEGylated lipid of structure (IV), such as compound IVa).In some embodiments, nucleoside modified RNA is encapsulated in the lipid portion of lipid nanoparticle or the aqueous space that is covered by part or all of the lipid portion of lipid nanoparticle, thereby protecting it from enzyme cleavage or other undesirable effects induced by host organism or cellular mechanisms, such as harmful immune response.

[0308] In various embodiments, the lipid nanoparticles may be 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 have an average diameter of 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 nucleoside modified RNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by nucleases.

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

[0310] In one embodiment, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids. The stabilizing lipids include neutral lipids and PEGylated lipids.

[0311] In one embodiment, LNP comprises cationic lipid.As used herein, the term "cationic lipid" refers to lipid that is cationic or becomes cationic (protonated) as the pH is lower than the pK of the ionic group of the lipid, but is gradually more neutral at higher pH values.At pH values ​​below pK, the lipid can then bind to negatively charged nucleic acid.In some embodiments, cationic lipid comprises zwitterionic lipid, which assumes positive charge as pH decreases.

[0312] In certain embodiments, the cationic lipid comprises any of a number of lipid spaces that have a net positive charge at a selected pH, such as physiological pH. Such lipids include 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 ... Cationic lipids include, but are not limited to, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), ... These include, for example, LIPOFECTIN® (cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), available from GIBCO / BRL, Grand Island, NY); LIPOFECT Amine® (cationic liposomes containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), available from GIBCO / BRL); and TRANSFECTAM® (cationic lipids containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, available from Promega Corp., Madison, Wis.).The following lipids are cationic, carrying a positive charge below physiological pH:

[0313] DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-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 WO 2012 / 016184, the entire contents of which are incorporated herein by reference.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).

[0315] In certain embodiments, the cationic lipid is present in the LNP in an amount of about 30 to about 95 molar percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 molar percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 to about 60 molar percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 molar percent. In one embodiment, the LNP comprises only cationic lipid. In certain embodiments, the LNP comprises one or more additional lipids that stabilize the formation of the particles 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 at physiological pH in either an uncharged or neutral zwitterionic form.

[0318] Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0319] Exemplary neutral lipids are, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), dipalmitoyloleoylphosphatidylcholine (POPC), dipalmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine. Include 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-stearyl-2-oleoyl-phosphatidiethanolamine (SOPE), and 1,2-dielideyl-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 LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipid (e.g., lipid of formula (I)) to neutral lipid ranges from about 2:1 to about 8:1.

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

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

[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, palmitoyloleolphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

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

[0325] In some embodiments, the LNP comprises a polymer-linked lipid. The term "polymer-linked lipid" refers to a molecule that comprises both a lipid portion and a polymer portion. An example of a polymer-linked lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethylmethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-s-DMG) and the like.

[0326] In certain embodiments, the LNPs include an additional stabilizing lipid that is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol lipids include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

[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 LNPs are PEGylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols, such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(co-methoxy(polyethoxy)ethyl)butanediol (PEG-S-DMG). PEGylated lipids include PEG-S-DAG, PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates 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 cationic lipid to 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 LNPs comprise 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 internalization domains. For example, in one embodiment, the LNP comprises one or more domains that bind to cells and induce internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to receptors found on the fat surface and induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding to a molecule in vivo, where the LNP-bound molecule can then be recognized by a cell surface receptor and induce internalization. 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 production are described in, e.g., WO 2016176330A1, U.S. Patent Publication No. US20120276209, 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 acid. 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 by reference in their entirety.

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

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

[0334] In another embodiment, the pharmaceutical compositions provided herein are controlled release compositions, i.e., compositions in which the compound is released over an extended period of time after administration. Controlled or sustained release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the compositions are immediate release compositions, i.e., compositions in which the entire compound is released immediately after 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 invention provides kits containing reagents utilized in practicing the methods of the invention. In another embodiment, the invention provides kits containing the compositions, tools, or instructions of the invention.

[0337] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention but should not be construed in any way as limiting the broad scope of the invention. EXAMPLES

[0338] Example 1: Materials and Experimental Methods Modified mRNAs expressing HSV-2 glycoproteins C, D and E (gC2 / gD2 / gE2) ectodomains. 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 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 liposomal nanoparticles (LNPs) by Acuitas Therapeutics to prepare the following immunogens: (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 and gD2 and gE2 modified mRNA in LNP.

[0340] The immunization groups were as follows: a) Control (polyvalent C group): Polyvalent C mRNA / LNP divided into four aliquots and administered at four separate sites. b) gD2 alone (group gD2): 10 μg of gD2 mRNA / LNP administered in four aliquots at four separate sites. c) Individual trivalents (Trivalent-I group): gC2mRNA / LNP 10 μg, gD2mRNA / LNP 10 μg, gE2mRNA / LNP 10 μg, each divided into two aliquots and administered at two sites each. d) Combined trivalent (Trivalent-C group): 10 μg gC2 mRNA, 10 μg gD2 mRNA and 10 μg gE2 mRNA combined in LNP, divided into four aliquots and administered at four separate sites.

[0341] Experimental procedure. Hair was removed from the backs of 6-8 week old BALB / c mice using an electric laser and a Nile. Mice were bled prior to the first and second immunizations and prior to intravaginal challenge. Two immunizations were performed intradermally, 28 days apart. Intradermal immunizations were performed on the exposed back. Five mice (group c above) that had received the trivalent vaccine at individual sites were sacrificed 14 days after the second immunization. CD4 + and CD8 + Spleens were harvested for T cell responses. 28 days after the second immunization, mice were treated subcutaneously with 2 mg of Depo-Provera, and 5 days later with 5 × 10 3 PFUHSV-2 strain MS (approximately 400LD 50 Mice were intravaginally infected with 1000 sera from vaccinated groups. At 2 and 4 days post-challenge, vaginal swabs were obtained for viral culture. At 4 days post-challenge, several mice in each vaccinated group were sacrificed and dorsal root ganglia (DRG) were excised for HSV-2 DNA qPCR. The remaining animals were evaluated for weight loss and hind limb weakness for 10 days, while survival and reproductive disease were monitored for 28 days.

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

[0343] Conclusions: When transfected into mammalian cells, modified mRNAs encoding the ectodomains of HSV-2 gC2 (Fig. 1A), gD2 (Fig. 1B), and gE2 (Fig. 1C) produced appropriate molecular proteins that reacted with antibodies against the glycoproteins in Western blots.

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

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

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

[0347] Example 4: Balanced T produced by modified mRNA immunization H 1 and T H 2 IgG isotype T H 1 or T H 2 The ability of mRNA immunization to primarily stimulate immune responses was examined using IgG1 (T H 2) or IgG2a(T H1) Antibodies were produced by determining whether the antigens produced antibodies. ELISA was performed on plates coated with all three antigens gC2, gD2 and gE2. Sera obtained after the first or second immunization were added to the antigen-coated plates, and IgG1 or IgG2a was detected using HRP anti-mouse IgG1 or IgG2a. IgG1 (Figure 3A) and IgG2a (Figure 3B) titers were significantly elevated after immunization with gD2 and trivalent modified mRNA vaccination. Furthermore, 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 show that titers of antibodies against both IgG1 and IgG2a isotypes were generated, indicating a balanced titer response to immunization with modified gC2, gD2 and gE2 mRNA. H 1 and T H Two responses are shown.

[0349] Example 5: High neutralizing antibody titers following modified MRNA immunization Serum was obtained 28 days after the second immunization 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 individuals seronegative for HSV-1 and HSV-2. Each of the modified mRNA groups was significantly different from the polyvalent C control (p<0.001; FIG. 4). While each of the mRNA groups was not significantly different from each other, trivalent vaccination administered as a combined immunogen (trivalent-C) performed as the best of the three mRNA groups (FIG. 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 in splenocytes after modified MRNA immunization + and CD8 + T cell response Five animals in the trivalent modified mRNA group (Trivalent-I group) immunized with each glycoprotein mRNA at separate sites were euthanized 14 days after the second immunization. Splenocytes were prepared for T cell assays. Splenocytes were stimulated with glycoprotein subunit antigens or 15 amino acid peptides containing 11 overlapping amino acids prepared in baculovirus. CD4 + and CD8 + The T cell responses are shown in Figures 5 and 6, respectively.

[0352] CD4 + T cells: Modified mRNA expressing gC2, gD2, and gE2 subunit antigens, respectively, induces polyfunctional CD4 + Spleen cells harvested from immunized subjects and then stimulated with the subunit antigen glycoprotein stimulated polyfunctional CD4 T cell responses (Figures 5A-5B). + Splenocytes harvested from immunized subjects and then stimulated with the 15 amino acid overlapping peptides elicited polyfunctional CD4 T cell responses (Figure 5A). + Increased T cell and IFNγ responses (Figure 5B). CD8 + T cells: Only gE peptide pool 2 significantly + stimulated a T cell response (Figure 6B).

[0353] Example 7: Survival, weight loss and neurological signs following modified mRNA immunization and intravaginal challenge Thirty-three days after the second immunization, animals were administered 5 × 10 3 PFU of HSV-2 strain MS (approximately 400LD 50) was inoculated intravaginally. Animals were observed daily for survival, neurological signs consisting of hind limb weakness or paralysis and hunched gait, and weight loss or gain. All animals in the polyvalent C control group died, whereas all animals in gD2 alone, trivalent administered individually (labeled trivalent-I) or trivalent administered in combination (labeled trivalent-C) survived (Figure 7A; p=0.002 by Log-rank (Mantel-Cox) comparing the three mRNA / LNP groups with the polyvalent C control). Figure 7B shows that administration of the modified mRNA vaccine twice at an interval of 28 days and intravaginal challenge with HSV-2 did not result in neurological signs or weight loss. Control subjects administered the vaccine 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 were able to immunize with approximately 400 LD of HSV-2. 50 After intravaginal challenge with 100 mg of ...

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

[0356] Each of the mRNA / LNP groups was significantly superior to the polyvalent C control group. Notably, vaginal titers on days 2 and 4 post-challenge were negative in mice immunized with trivalent mRNA, whether administered at separate sites or as a combined immunization. Comparing either trivalent group to gD2 alone did not detect significant differences, however, both trivalent groups were superior to the gD2 alone group, as 3 out of 10 mice in the gD2 group had virus isolated from vaginal swabs.

[0357] Example 9: Genital Disease Following Modified mRNA Immunization and Intravaginal Challenge Animals were monitored daily for genital disease for 28 days after challenge, with a score of 0 designated as no disease and 1 point designated as loss of hair around the anus or genital opening, genital erythema, genital exudate, and necrosis of reproductive tissue (Figure 9).

[0358] No animals in the gD2 or trivalent mRNA / LNP groups developed reproductive disease and were significantly different from the polyvalent C controls (p<0.001, one-way ANOVA with Kruskal-Wallis test followed by Dunn's multiple comparisons for significance).

[0359] Example 10: HSV-2 DNA in dorsal root ganglia following modified MRNA immunization and intravaginal challenge Five animals per group were euthanized 4 days after challenge, except for the trivalent combined group, in which four animals were euthanized. Dorsal root ganglia (DRG) were harvested for HSV-2 DNA quantification by qPCR to detect the Us9 gene. All five animals in the polyvalent C group had HSV-2 DNA detected in the DRG, whereas 2 of 5 animals in gD mRNA, 1 of 5 animals in trivalent mRNA at individual sites, and 1 of 4 animals administered trivalent mRNA at the same site were positive for HSV-2 DNA (Figure 10; Mann-Whitney test: gD2 compared to polyvalent C, p=0.03; trivalent at different sites compared to polyvalent C, p<0.01; trivalent at same site compared to polyvalent C, p=0.14). Differences between groups immunized with modified mRNAs were not significant.

[0360] Conclusion: Dorsal root ganglia were positive for HSV-2 DNA at day 4 postinfection in 75%-80% of animals immunized with gD2 alone or the trivalent vaccine. The trivalent mRNA and gD2 mRNA groups at different sites were significantly superior to the multivalent C mRNA control group, whereas the trivalent mRNA group containing all glycoproteins administered together was not significantly different from the multivalent C group, possibly due to the smaller sample size in the trivalent combination group.

[0361] overview Modified mRNA vaccines expressing gD2 alone or gC2, gD2 and gE2 provided outstanding protection against HSV-2 genital challenge. Expression of the three proteins was significantly superior to gD2 based on titers at days 2 and 4 post-challenge, and fewer animals with HSV-2 DNA were detected in the DRG at day 4.

[0362] Example 11: Follicular helper T (Tfh) cell and germinal center B cell responses in immunized mice BALB / c female mice were left unimmunized as negative control animals or immunized intradermally with multivalent CmRNA-LNP or valence-modified mRNA-LNP at 28-day intervals. Multivalent CmRNA controls received 10 μg of multivalent CmRNA-LNP divided into four aliquots and administered at four separate sites. Trivalent modified mRNA groups received 10 μg of gC2mRNA-LNP, 10 μg of gD2mRNA-LNP, and 10 μg of gE2mRNA-LNP, each divided into two aliquots and administered at two sites each. Two weeks after the second immunization, spleens were harvested from five animals per group and flow cytometry was performed to determine the abundance of follicular helper T (Tfh) cells (FIG. 11A; * p<0.05) and germinal center B cell responses ( Fig. 11B ; * p<0.05) was detected.

[0363] Conclusion: The trivalent mRNA-LNP vaccine induced strong Tfh and germinal center B cell responses, which were significantly superior to the polyvalent C control immunization (p<0.05) and negative groups (p<0.05) for both Tfh and germinal center B cell responses. These immune responses suggest that the trivalent modified mRNA-LNP vaccine likely induces durable antibody responses.

[0364] Example 12: Vaginal IgG responses to modified MRNA immunization in mice BALB / c mice were immunized intradermally twice with 10 μg of multivalent CmRNA-LNP, 10 μg of gD2mRNA-LNP, or 10 μg of trivalent modified mRNA-LNPs of gC2, gD2, and gE, at an interval of 28 days. Trivalent mRNAs were combined and combined into LNPs, and administered at four sites: 10 μg of gC2mRNA, 10 μg of gD2mRNA, and 10 μg of gE2mRNA. One month after the second immunization, 60 μl of medium was introduced into the vaginal cavity and allowed to recover. IgG titers against gC2 (Figure 12A), gD2 (Figure 12B), and gE2 (Figure 12C) were determined by ELISA in vaginal washes at 1:50 dilution (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 generated strong vaginal IgG responses to gC2 (Figure 12A) and gD2 (Figure 12B) and more moderate responses to gE2 (Figure 12C). gD2 ELISA titers were 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 generated by trivalent MRNA immunization of mice block immune evasion domains on gC2 BALB / c mice were left unimmunized or immunized intradermally with multivalent CmRNA-LNP or trivalent mRNA-LNP as a source of non-immune IgG. The multivalent CmRNA control received 10 μg of multivalent CmRNA-LNP, divided into four aliquots and administered at four separate sites. 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 into one LNP, divided into four aliquots, and administered at four sites. There were 10 mice in each group. Serum from the 10 mice was pooled and IgG was purified. IgG was evaluated at 12 μg / 200 μl for its ability to block complement component C3b binding to gC2. This blocking assay is used to evaluate whether antibodies generated by immunization block the immune evasive properties of gC2. Non-immunized mouse IgG, IgG from the multivalent CmRNA group, and IgG from the gD2mRNA group, respectively, did not block gC2 binding to C3b. In contrast, IgG from mice immunized with trivalent mRNA totally blocked the interaction between gC2 and C3b (FIG. 13, **** p<0.0001).

[0367] Conclusions: The trivalent mRNA vaccine generates antibodies that block immune evasion domains on gC2, as determined by blocking the interaction between gC2 and C3b.

[0368] Example 14: Intravaginal infection of mice with higher titers of HSV-2 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, each divided into two aliquots and administered individually at two sites. One month after the second immunization, mice were treated with medroxyprogesterone and 5 days later, 5×104 PFU HSV-2 strain MS (2,000LD 50 ) were infected intravaginally. Animals were followed for 28 days and evaluated for mortality, genital disease, vaginal viral titers 2 and 4 days post-infection, and dorsal root ganglion (DRG) HSV-2 DNA copy numbers 28 days post-infection. Mice immunized with the trivalent mRNA-LNP vaccine had no mortality, no genital disease, no virus detected 2 or 4 days post-infection, or no HSV-2 DNA detected in the DRG (Table 1). [Table 1]

[0369] Conclusion: Mice were infected with a 10-fold higher dose of HSV-2 than used in the previous experiments described herein (Figures 7-10). Protection of mice remained outstanding even at this higher titer challenge. We achieved stable immunity in all five mice as determined by no deaths at 2 and 4 days post-infection, no genital disease, negative vaginal viral titers, 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 with polyvalent C mRNA-LNPs as a control (15 / group) or with trivalent mRNAs containing 10 μg each of gC2, gD2, and gE2 mRNA-LNPs (20 / group). All polyvalent C control animals died by day 12, whereas all animals in the trivalent mRNA group survived (Figure 14A). No weight loss occurred in the trivalent mRNA group, whereas polyvalent C control animals lost >15% of their body weight (Figure 14B). The polyvalent C group developed extensive genital disease, whereas the trivalent mRNA animals had no genital disease (Figure 14C). DRGs were collected from nine polyvalent C animals at the time of euthanasia 7-12 days post-infection, or at the end of the experiment on day 28 in the trivalent mRNA group. All animals in the polyvalent C group had HSV-2 DNA detected in the DRG, whereas none in the trivalent mRNA group were positive for HSV-2 DNA (Figure 14D). Vaginal viral cultures on days 2 (Figure 14E) and 4 (Figure 14F) were positive in all 15 animals in the polyvalent C group, whereas cultures were negative in all 20 animals in the trivalent mRNA group. The difference between the polyvalent C and trivalent groups was significant, p<0.001 for each figure (Figures 14A-14F).

[0371] Conclusion: Trivalent modified mRNA-LNPs provide outstanding protection in mice when administered intramuscularly. We reported comparable findings above when mice were immunized intradermally. Overall, we evaluated here 64 mice immunized with 10 μg of trivalent mRNA of each immunogen administered either intradermally (Figures 7-10) or intramuscularly (Figure 14). We achieved stabilized immunity in 63 / 64 (98%) mice based on no deaths, no reproductive disease, no weight loss, negative day 2 and 4 vaginal titers and negative HSV-2 DNA in DRG.

[0372] Example 16: Summary of comparison of immunization with trivalent mRNA-LNP and trivalent subunit antigen CPG / alum in BALB / c mice The results presented in Table 2 herein below represent an overview of all results in BALB / c mice immunized either intradermally or intramuscularly with trivalent mRNA containing 10 μg each of gC2, gD2 and gE2 mRNA-LNP (a total of 64 mice were studied). We 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. gC2, gD2, gE2 subunit antigens were mixed with 150 μg CpG and 25 μg alum per μg protein as adjuvants and administered intramuscularly. As we did in previous experiments, mice were immunized with trivalent mRNA-LNP twice at 28-day intervals and with subunit antigens three times at 14-day intervals. The mRNA and subunit antigen experiments were performed simultaneously. The results summarized in Table 2 show the 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 stable 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 unimmunized and uninfected (negative group, n=10) and were immunized intradermally three times at one-month intervals with 20 μg of polyvalent C mRNA-LNPs (n=10) or 20 μg each of gC2, gD2, and gE modified mRNA-LNPs (n=10). One month after the final immunization, animals in the polyvalent C and trivalent mRNA groups were administered 5×10 5 PFU of HSV-2 strain MS (50LD 50 ) were intravaginally infected with genital lesions during the acute phase of infection (days 1-14) and during the relapse phase of infection (days 15-60). Animals were observed for death, genital lesions during the acute phase of infection (days 1-14), and genital lesions during the relapse phase of infection (days 15-60). In the polyvalent C control group, 7 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 (uninfected) animals died (Figure 15A). The polyvalent C group had genital lesions at a median of 6.4 days during the acute phase of infection, including 9 of 10 animals developing acute genital disease, while none of the animals in the trivalent or untreated (uninfected) groups developed acute genital disease (Figure 15B). The polyvalent C animals had genital lesions at a median of 3.7 days during the relapse phase of infection, including 2 of 3 animals developing relapsed genital lesions (Figure 15C). In contrast, trivalent immunized guinea pigs and naive (uninfected) animals did not have recurrent genital lesions (FIG. 15C). Conclusions: Trivalent modified mRNA-LNPs conferred 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, and it should be understood that the present invention is not limited to the precise 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 present invention as defined in the appended claims.

[0375] All patent documents and references cited herein are incorporated by reference as if fully set forth. The present invention provides, for example, the following items. (Item 1) A composition comprising one or more nucleoside modified mRNAs, each of which encodes a herpes simplex virus (HSV) glycoprotein or an immunogenic fragment thereof, and wherein the nucleoside modified mRNAs comprise one or more pseudouridine residues. (Item 2) 2. The composition of claim 1, wherein the one or more pseudouridine residues include mlψ (1-methylpseudouridine). (Item 3) The one or more pseudouridine residues are 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. (Item 4) 4. The composition of any one of items 1 to 3, wherein the one or more nucleoside modified mRNAs encode a) HSV glycoprotein D (gD) or an immunogenic fragment thereof, b) HSV glycoprotein C (gC) or an immunogenic fragment thereof, and c) HSV glycoprotein E (gE) or an immunogenic fragment thereof, or any combination thereof. (Item 5) 5. The composition of item 4, wherein the HSV glycoprotein comprises an HSV-1 glycoprotein. (Item 6) 5. The composition of item 4, wherein the HSV glycoprotein comprises an HSV-2 glycoprotein. (Item 7) 7. The composition according to item 6, wherein the nucleoside modified mRNA encoding the immunogenic fragment of gD immunogenic fragment of HSV comprises amino acids 26 to 331 from HSV-2 strain 333 or a homologous sequence from another HSV strain. (Item 8) 8. The composition of item 7, wherein the nucleic acid sequence of the nucleoside modified mRNA is as set forth in SEQ ID NO:4. (Item 9) 9. The composition of any one of items 4 to 8, wherein the immunogenic fragment of HSV gC comprises either the C3b-binding domain thereof, the properdin-interfering domain thereof, the C5-interfering domain thereof, or a fragment of the C3b-binding domain, the properdin-interfering domain, or the C5-interfering domain. (Item 10) 10. The composition according to any one of items 6 to 9, wherein 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. (Item 11) 11. The composition of claim 10, wherein the nucleic acid sequence of the nucleoside modified mRNA is as set forth in SEQ ID NO: 10. (Item 12) 12. The composition according to any one of items 6 to 11, wherein 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. (Item 13) 13. The composition of claim 12, wherein the nucleic acid sequence of the nucleoside modified mRNA is as set forth in SEQ ID NO: 16. (Item 14) 14. The composition of any one of items 1 to 13, wherein the one or more 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. (Item 15) 15. The composition of any one of items 1 to 14, wherein one or more of the nucleoside modified mRNAs further comprises a poly A tail. (Item 16) 16. The composition of any one of items 1 to 15, wherein one or more of the nucleoside modified mRNAs further comprises an m7GpppG cap, a 3'-O-methyl-m7GpppG cap, or an anti-reverse cap analog. (Item 17) 17. The composition of any one of items 1 to 16, wherein one or more of the nucleoside modified mRNAs further comprises a cap-independent translation enhancer. (Item 18) 18. The composition of any one of items 1 to 17, wherein one or more of the nucleoside modified mRNAs further comprise 5' and 3' untranslated regions that facilitate translation. (Item 19) 19. The composition of any one of items 1 to 18, wherein one or more of the nucleoside modified mRNAs are encapsulated in a nanoparticle, lipid, polymer, cholesterol, or a cell-penetrating peptide. (Item 20) 20. The composition of claim 19, wherein the nanoparticle is a liposomal nanoparticle. (Item 21) 21. A method of treating a herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition of nucleoside modified mRNA according to any one of items 1 to 20. (Item 22) 21. A method of suppressing, inhibiting, or reducing an incidence of herpes simplex virus (HSV) infection in a subject, comprising administering to the subject a composition of nucleoside modified mRNA according to any one of items 1 to 20. (Item 23) 23. The method of any one of items 21 to 22, wherein the HSV infection comprises an HSV-1 infection. (Item 24) 23. The method of any one of items 21 to 22, wherein the HSV infection comprises an HSV-2 infection. (Item 25) 25. The method of any one of items 21 to 24, wherein the HSV infection comprises a primary HSV infection. (Item 26) The HSV infection is secondary to a primary HSV infection. Relapse 25. The method according to any one of items 21 to 24, comprising treating with HSV labialis, recurrent HSV or oral HSV. (Item 27) 25. The method of any one of items 21 to 24, wherein the HSV infection comprises reactivation of a latent HSV infection. (Item 28) 28. The method of any one of items 21 to 27, wherein the HSV infection comprises HSV encephalitis, HSV neonatal infection, genital HSV infection, or oral HSV infection. (Item 29) 21. 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) 30. The method of any one of items 21 to 29, wherein the administering step comprises intramuscular administration. (Item 31) 30. The method of any one of items 21 to 29, wherein the administering step comprises subcutaneous administration. (Item 32) 30. The method of any one of items 21 to 29, wherein the administering step comprises intradermal administration. (Item 33) 30. The method of any one of items 21 to 29, wherein the administering step comprises intranasal, intravaginal, or intrarectal administration. (Item 34) 30. The method of any one of items 21 to 29, wherein the administering step comprises topical administration. (Item 35) 35. The method of any one of items 21 to 34, wherein the administering step comprises a) administering a first composition comprising a nucleoside-modified mRNA encoding a first HSV glycoprotein, b) administering a second composition comprising a nucleoside-modified mRNA encoding a second HSV glycoprotein, and c) administering a third composition comprising a nucleoside-modified mRNA encoding a third HSV glycoprotein. (Item 36) 36. The method of claim 35, wherein the first composition, the second composition, and the third composition are administered to the subject at a single administration site. (Item 37) 36. The method of claim 35, wherein the first composition, the second composition, and the third composition are administered to the subject at different administration sites. (Item 38) 38. The method of any one of items 35 to 37, wherein the first composition, the second composition, and the third composition are administered simultaneously. (Item 39) 38. The method of any one of items 35 to 37, wherein the first composition, the second composition, and the third composition are administered sequentially. (Item 40) 40. The method of any one of items 35 to 39, wherein the first composition, the second composition, and the third composition are administered by the same route of administration. (Item 41) 40. The method of any one of items 35 to 39, wherein the first composition, the second composition, and the third composition are administered by different routes of administration. (Item 42) 42. The method of any one of items 21 to 41, further comprising administering to the subject one or more additional doses of the nucleoside modified mRNA composition after the first administration. (Item 43) 42. The method of any one of items 21 to 41, further comprising the step of administering to the subject a composition comprising the HSV glycoprotein or an immunogenic fragment thereof. (Item 44) 44. The method of claim 43, wherein the composition comprising the HSV glycoprotein is administered following administration of the nucleoside modified mRNA composition. (Item 45) 44. The method of claim 43, wherein the composition comprising the HSV glycoprotein is administered prior to administration of the composition of nucleoside modified mRNA. (Item 46) 46. ​​The method of any one of items 43 to 45, wherein the HSV glycoprotein comprises gD, gC, gE or a combination thereof. (Item 47) 47. The method of any one of items 29 to 46, wherein the immune response comprises a CD4 immune response. (Item 48) 47. The method of any one of items 29 to 46, wherein the immune response comprises a CD8 immune response. (Item 49) 47. The method of any one of items 29 to 46, wherein the immune response comprises a follicular helper T cell immune response. (Item 50) 47. The method of any one of items 29 to 46, wherein the immune response comprises a germinal center B cell immune response. (Item 51) 47. The method of any one of items 29 to 46, wherein the immune response comprises an IgG antibody response against gC2, gD2, gE2, or a combination thereof.

Claims

1. (i) a composition for treating herpes simplex virus (HSV) infection or suppressing, inhibiting or reducing the incidence of HSV infection, or (ii) for eliciting an immune response, comprising: wherein the composition comprises: (a) an RNA encoding an ectodomain of herpes simplex virus (HSV) glycoprotein D (gD) comprising a sequence that is 90%-97% identical to SEQ ID NO:5; (b) an RNA encoding an ectodomain of HSV glycoprotein C (gC) comprising a sequence that is 90%-97% identical to SEQ ID NO:11; and (c) an RNA encoding an ectodomain of HSV glycoprotein E (gE) comprising a sequence that is 90%-97% identical to SEQ ID NO:17; wherein one or more of said RNAs is nucleoside modified RNA.

2. The composition of claim 1, wherein the RNA encoding the ectodomain of HSV gD comprises a sequence that is 95% to 97% identical to SEQ ID NO:5; the RNA encoding the ectodomain of HSV gC comprises a sequence that is 95% to 97% identical to SEQ ID NO:11; and the RNA encoding the ectodomain of HSV gE comprises a sequence that is 95% to 97% identical to SEQ ID NO:

17.

3. The composition of claim 1 , wherein the nucleoside modified RNA comprises one or more pseudouridine residues.

4. 4. The composition of claim 3, wherein the one or more pseudouridine residues comprise mlψ (1-methylpseudouridine), mlacp3ψ (1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine), ψm (2'-O-methylpseudouridine), m5D (5-methyldihydrouridine), m3ψ (3-methylpseudouridine), or any combination thereof.

5. The composition of claim 1, further comprising one or more RNAs encoding 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, or d) HSV glycoprotein I (gI), or e) any combination thereof.

6. The composition of claim 5, wherein one or more of the glycoproteins are from HSV-1.

7. The composition of claim 5, wherein one or more of the glycoproteins are from HSV-2.

8. The RNA is i) a polyA tail; ii) m7GpppG cap, 3'-O-methyl-m7GpppG cap, or anti-reverse cap analog; iii) cap-independent translation enhancers; iv) 5' and 3' untranslated regions that facilitate translation; or v) Combinations of these The composition of claim 1 further comprising:

9. The composition of claim 1 , wherein the RNA is encapsulated in a nanoparticle, lipid, polymer, cholesterol, or a cell-penetrating peptide.

10. The composition of claim 9 , wherein the nanoparticle is a liposomal nanoparticle.

11. 2. The composition of claim 1, wherein the RNA encoding the ectodomain of the HSV gE consists of a nucleotide sequence having at least 75% identity to nucleotides 200-1348 of SEQ ID NO:

16.

12. The composition of claim 1 , wherein one or more of the nucleoside modified RNAs further comprises a signal sequence.

13. The signal sequence is a. AUGACCCGCCUGACCGUGCUGGCCCUGCUGGCCGGCCUGCUGGCCUCCUCCCGCGCC (SEQ ID NO: 19), b. AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACUCC (SEQ ID NO: 20), or c. AUGGCCAUCUCCGGCGUGCCCGUGCUGGGCUUCUUCAUCAUCGCCGUGCUGAUGUCGCCCAGGAGUCCUGGGCC (SEQ ID NO: 21) The composition of claim 12 comprising:

14. A composition according to any one of claims 1 to 13 for treating herpes simplex virus (HSV) infection or for suppressing, inhibiting or reducing the incidence of HSV infection.

15. The composition of claim 14, wherein the HSV infection comprises an HSV-1 infection or an HSV-2 infection.

16. 15. The composition of claim 14, wherein the HSV infection comprises a primary HSV infection; a relapse, recurrence, or oral HSV following a primary HSV infection; a reactivation of a latent HSV infection; or HSV encephalitis, HSV neonatal infection, genital HSV infection, or oral HSV infection.

17. 15. The composition of claim 14, wherein the composition is formulated for intramuscular, subcutaneous, intradermal, intranasal, intravaginal, intrarectal, or topical administration.

18. The composition according to any one of claims 1 to 13 for inducing an immune response.

19. 20. The composition of claim 18, 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; an IgG antibody response against gC2, gD2, gE2, or a combination thereof; or a combination thereof.

20. 20. The composition of claim 18, wherein the composition is formulated for intramuscular, subcutaneous, intradermal, intranasal, intravaginal, intrarectal, or topical administration.

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