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WO2025030165A1 - Compositions pharmaceutiques pour l'administration d'antigènes du virus de l'herpès simplex et procédés associés - Google Patents

Compositions pharmaceutiques pour l'administration d'antigènes du virus de l'herpès simplex et procédés associés Download PDF

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Publication number
WO2025030165A1
WO2025030165A1 PCT/US2024/040874 US2024040874W WO2025030165A1 WO 2025030165 A1 WO2025030165 A1 WO 2025030165A1 US 2024040874 W US2024040874 W US 2024040874W WO 2025030165 A1 WO2025030165 A1 WO 2025030165A1
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Prior art keywords
hsv
polypeptides
antigenic
antigenic fragments
polypeptide
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Inventor
Alptekin GÜLER
Ricardo Sanchez Velazquez
Julia UEBELE
Annette VOGEL
Christina PFAFENROT
Anna Luise Ernst
Stephanie HEIN
Sabrina Hinz
Ugur Sahin
Michael Steven ROONEY
Ekaterina ESAULOVA
Adam ZUIANI
Theresa ADDONA
Asaf PORAN
Scott GOULDING
Alexandra WALLS
John SROUJI
Gavin PALOWITCH
Charles Lefco DULBERGER
Sarah Catharina Dany
Stefan Thomas STRAUSS
Gary H. Cohen
Harvey Friedman
Sita Awasthi
Drew Weissman
Lauren Michelle HOOK
Kevin EGAN
Tina M. Cairns
Doina ATANASIU
Wan Ting SAW
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Biontech SE
University of Pennsylvania Penn
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Biontech SE
University of Pennsylvania Penn
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Publication of WO2025030165A1 publication Critical patent/WO2025030165A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Herpes simplex viruses commonly referred to only as herpes, are categorized into two types: herpes simplex virus, type 1 (HSV-1, or oral herpes) and herpes simplex virus, type 2 (HSV-2, or genital herpes).
  • HSV-1 herpes simplex virus
  • HSV-2 herpes simplex virus
  • HSV-1 type 1
  • HSV-2 herpes simplex virus
  • HSV-1 prevalence is understood as being highest in Africa and lowest in the Americas.
  • HSV-2 More women are infected with HSV-2 than men, because sexual transmission of HSV is more efficient from men to women than from women to men. Prevalence of HSV-2 infection was estimated to be highest in Africa, followed by the Americas. Prevalence of HSV-2 was also shown to increase with age, though the highest numbers of people newly-infected have historically been in adolescents. Both HSV-1 and HSV-2 infections are lifelong.
  • technologies e.g., combinations, compositions, methods, etc.
  • the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) for delivering herpes simplex virus (HSV) antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods).
  • pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • HSV compositions e.g., immunogenic compositions, e.g., vaccines
  • related technologies e.g., methods.
  • HSV glycoprotein C or antigenic portions thereof, HSV glycoprotein D (gD) or antigenic portions thereof, glycoprotein E (gE) or antigenic portions thereof, or combinations thereof can be useful in preventing or treating HSV, e.g., HSV-1, HSV-2, or both, as further disclosed herein.
  • the present disclosure provides, for example, combinations comprising a plurality of polyribonucleotides, wherein the plurality of polyribonucleotides comprises a first set of polyribonucleotides that encode one or more glycoprotein (GP) polypeptides.
  • GP polypeptide comprises an HSV glycoprotein or antigenic portions thereof.
  • polyribonucleotides that encode one or more GP polypeptides provided herein encode one or more of HSV-2 gC, gD, and/or gE or antigenic portions thereof (e.g., in a construct).
  • such polyribonucleotides of a first set of polyribonucleotides can be part of an RNA construct.
  • a polyribonucleotide that encodes a GP polypeptide or RNA construct as described herein can be part of a composition (e.g., a pharmaceutical composition, e.g., an immunogenic composition, e.g., a vaccine).
  • the present disclosure also provides a combination comprising a plurality of polyribonucleotides, wherein the plurality of polyribonucleotides comprises a first set of polyribonucleotides and a second set of polyribonucleotides.
  • a second set of polyribonucleotides encode one or more T-cell string polypeptides.
  • a T-cell string polypeptide comprises one or more HSV T-cell antigens or antigenic portions thereof.
  • such polyribonucleotides of a second set of polyribonucleotides can be part of an RNA construct.
  • a polyribonucleotide that encodes a T-cell string polypeptide or a corresponding RNA construct as described herein can be part of a composition (e.g., a pharmaceutical composition, e.g., an immunogenic composition, e.g., a vaccine).
  • a composition e.g., a pharmaceutical composition, e.g., an immunogenic composition, e.g., a vaccine.
  • FIG.2 has been modified from Ibanez, F.J., et al., “Experimental Dissection of the Lytic Replication Cycles of Herpes Simplex Virus in vitro,” Front Microbiol. 2018; 9: 2406, which is incorporated herein by reference in its entirety.
  • FIGS.3A-3F show HSV-2 gC, gD, or gE expression in HEK293T cells transfected with 0.2 ⁇ g/mL LNP-formulated trivalent nucleoside-modified RNA (modRNA) encoding gC, gD, and gE (drug product, DP).
  • modRNA trivalent nucleoside-modified RNA
  • HSV-2 gC, gD and gE protein were detected by flow cytometry using primary monoclonal mouse antibodies detecting the respective antigen and a secondary fluorescent tagged anti-mouse antibody.
  • Representative data from one experiment showing percentage of gC (FIG.3A), gD (FIG.3B), and gE (FIG.3C) protein-expressing cells and median fluorescence intensities (MFI) (FIG.3D, 3E, and 3F, respectively) of the total HEK293T population are depicted per antigen. Data shown are mean+SD of HEK293T transfections performed in triplicates.
  • FIG.4 shows a schematic overview of a study in guinea pigs investigating a composition candidate against HSV-2.
  • Guinea pigs were immunized IM on day 0 and day 28 with an HSV-2 composition candidate containing total HSV-2 gC/gD/gE RNA at a concentration of 3 ⁇ g, 15 ⁇ g, or PBS control, as outlined in Table 24.
  • the guinea pigs were challenged with a lethal dose of 5 x 10 5 PFU of HSV-2 strain MS (25-fold LD 50 ).
  • FIGS.5A-5C show serum IgG antibody titers observed one month after a 2nd immunization in guinea pigs immunized with an HSV-2 composition candidate described herein.
  • Serum antibody titers were determined by ELISA at day 56, 4 weeks after the second immunization with a composition (“trivalent vaccine”) comprising three polyribonucleotides encoding glycoprotein C (gC), glycoprotein D (gD) and glycoprotein E (gE), respectively.
  • the dose level represents total RNA content of three RNAs encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens in a 1:1:1 ratio.
  • Geometric mean ⁇ 95% CI and individual animal values are shown. P values were calculated by Kruskal-Wallis test.
  • gC2 glycoprotein C from herpes simplex virus-2
  • gD2 glycoprotein D from herpes simplex virus-2
  • gE2 glycoprotein E from herpes simplex virus-2
  • IgG immunoglobulin G
  • RNA ribonucleic acid
  • GMT geometric mean
  • CI confidence interval.
  • FIG.5A administration of the HSV-2 composition candidate induced high IgG antibody titers against each of HSV-2 gC (FIG.5A), HSV-2 gD (FIG.5B) and HSV-2 gE (FIG.5C), with a 15 ⁇ g dose inducing higher titers for gC and gD antigens than a 3 ⁇ g dose.
  • FIGS.6A-6C show vaginal IgG antibody titers in guinea pigs one month after a 2nd immunization with an HSV-2 modRNA composition described herein. Vaginal antibody titers were determined by ELISA at day 56, four weeks after the second immunization with a trivalent composition.
  • the dose level represents total RNA content of three RNAs encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens in a 1:1:1 ratio. Geometric mean ⁇ 95% CI and individual animal values are shown. P values were calculated by Kruskal-Wallis test.
  • gC2 glycoprotein C from herpes simplex virus-2
  • gD2 glycoprotein D from herpes simplex virus-2
  • gE2 glycoprotein E from herpes simplex virus- 2
  • IgG immunoglobulin G
  • RNA ribonucleic acid
  • GMT geometric mean
  • CI confidence interval.
  • FIG.6A shows serum neutralizing antibody titers to HSV-2 in guinea pigs one month after a 2nd immunization with an HSV-2 modified RNA (modRNA) composition described herein.
  • modified RNA modified RNA
  • Neutralizing antibody titers were determined using a serum HSV-2 plaque reduction assay and defined as highest dilution of serum with 5% human complement that reduced the number of HSV-2 plaques by 50%. Samples were collected at day 56, 4 weeks after the second immunization. The dose level represents total RNA content of three RNAs encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens in a 1:1:1 ratio. Geometric mean ⁇ 95% CI and individual animal values are shown. P values were calculated by Mann-Whitney test.
  • FIGS.8A-8C shows weight loss in guinea pigs administered an HSV-2 composition described herein, following HSV-2 viral challenge.
  • RNA ribonucleic acid
  • FIG.9 shows survival of guinea pigs immunized with an HSV-2 composition described herein, up to day 48 after HSV-2 viral challenge. Probability of survival of guinea pigs up to 48 days after lethal intravaginal challenge with HSV-2 at day 60, approximately one month after second immunization with 3 ⁇ g or 15 ⁇ g of trivalent a composition, or PBS.
  • the dose level represents total RNA content of three RNAs encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens in a 1:1:1 ratio.
  • FIGS.10A-10C show individual evaluation of genital disease in guinea pigs administered an RNA composition described herein, up to day 48 after challenge with a lethal intravaginal dose of HSV-2. Results at day 60, approximately one month after the second vaccination with an HSV-2 modified RNA (modRNA) composition are shown.
  • FIG.10A shows the mean number of days with genital disease during this period and
  • FIG.10B shows the mean severity of genital lesions of days with genital disease. Mean ⁇ SEM and individual animal values are shown.
  • FIG.10C shows the mean number of urinary retention days.
  • the dose level represents total RNA content of three RNAs encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens in a 1:1:1 ratio.
  • P values were calculated by Mann-Whitney test. Black circles with red outlines are associated with animals that succumbed after viral challenge in the PBS group.
  • HSV-2 herpes simplex virus-2
  • SEM standard error of the mean
  • PBS phosphate buffered saline
  • gC2 glycoprotein C from herpes simplex virus-2
  • gD2 glycoprotein D from herpes simplex virus-2
  • gE2 glycoprotein E from herpes simplex virus-2
  • RNA ribonucleic acid
  • FIG.11 shows cumulative disease score in guinea pigs administered an RNA composition described herein, up to day 48 after challenge with a lethal intravaginal dose of HSV-2. Results at day 60 are shown, approximately one month after the second vaccination. The mean number of days with genital disease per group is shown over the course of 48 days. The dose level represents total RNA content of three RNAs encoding for the respective gC2, gD2 and gE2 antigens in a 1:1:1 ratio.
  • HSV-2 herpes simplex virus-2
  • PBS phosphate buffered saline
  • gC2 glycoprotein C from herpes simplex virus-2
  • gD2 glycoprotein D from herpes simplex virus-2
  • gE2 glycoprotein E from herpes simplex virus-2
  • RNA ribonucleic acid.
  • FIG.11 administration of 3 ⁇ g of an HSV-2 composition significantly decreased mean cumulative disease days, and administration 15 ⁇ g decreased mean cumulative disease days further still.
  • FIGS.12A-12C show vaginal virus titers in guinea pigs administered an HSV-2 composition described herein, 2 and 4 days after viral challenge.
  • Vaginal HSV-2 titers were determined by plaque assay 2 days (FIG.12A) and 4 days (FIG.12B) after a lethal intravaginal challenge with HSV-2. Results are plotted as means ⁇ SEM and individual animal values. Mean days of genital shedding of HSV-2 DNA were analyzed by PCR and displayed in (FIG.12C). The dose level for the HSV-2 composition represents total RNA content of three RNAs in a 1:1:1 ratio encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens. P values were calculated by Kruskal-Wallis test.
  • FIGS.13A-13B show DNA copy numbers in DRG and spinal cord of guinea pigs administered an HSV-2 composition disclosed herein, on day 48 after viral challenge. DRG and spinal cord HSV-2 DNA copy numbers in guinea pigs on day 48 following viral challenge with a lethal intravaginal dose of HSV-2 were analyzed by qPCR.
  • HSV-2 genome copies in DRG (FIG.13A) and spinal cord (FIG.13B) relative to GAPDH expression at day 48 after viral challenge are shown for immunized animals. Mean ⁇ SEM and individual animal values are shown.
  • the dose level for the HSV-2 composition represents total RNA content of three RNAs in a 1:1:1 ratio encoding for the respective HSV-2 gC, HSV-2 gD and HSV-2 gE antigens. P values were calculated by Mann-Whitney test.
  • FIGS.14A-14I show transfection rates and expression levels in HEK293T cells transfected with RNA encoding HSV-2 gC (gC2), HSV-2 gD (gD2) and HSV-2 gE (gE2) antigens.
  • Cells were transfected with 0.2 ⁇ g/mL modRNA encoding antigens using a commercial transfection reagent. Expression of gC2, gD2 and gE2 protein was detected by flow cytometry using primary monoclonal mouse antibodies detecting the respective antigen and a secondary fluorescent tagged anti-mouse antibody. Representative data from one experiment showing percentage of gC2 (FIG.14A), gD2 (FIG.14D) and gE2 (FIG.14G) protein-expressing cells and median fluorescence intensities (MFI) (FIG.14B, FIG.14E, and FIG.14H respectively) of the total HEK293T population are depicted per antigen.
  • MFI median fluorescence intensities
  • 2138 HSV-2 gE secretory signal and HSV-2 gC antigen (version 4)
  • 2140 HSV-1 gD secretory signal and HSV-2 gC antigen (version 4)
  • 2141 HSV-1 gB secretory signal and HSV-2 gC antigen (version 4)
  • 1876 IL2 secretory signal and HSV-2 gC antigen (version 3).
  • HSV-2 gD secretory signal and HSV-2 gD antigen version 1
  • 1877 HSV-2 gD secretory signal and HSV-2 gD antigen (version 3)
  • 1659 HSV-2 gD secretory signal and HSV-2 gD antigen (version 2)
  • 1660 HSV-2 gD secretory signal and HSV-2 gE antigen (version 2)
  • 2143 HSV-1 gD secretory signal and HSV-2 gE antigen (version 4).
  • FIGS.15A-15F show transfection rates and expression levels in HEK293T cells transfected with RNA encoding HSV-2 gC (gC2), HSV-2 gD (gD2) and HSV-2 gE (gE2) antigens.
  • Cells were transfected with 0.2 ⁇ g/mL modRNA encoding antigens using a commercial transfection reagent.
  • Expression of gC2, gD2 and gE2 protein was detected by flow cytometry using primary monoclonal mouse antibodies detecting the respective antigen and a secondary fluorescent tagged anti-mouse antibody.
  • FIG.15A Representative data from one experiment showing percentage of gC2 (FIG.15A) and gE2 (FIG.15B) protein-expressing cells and median fluorescence intensities of gC2, gD2 and gE2 (MFI) (FIG.15C, FIG.15D, and FIG.15E respectively) of the total HEK293T population are depicted per antigen. Data shown are mean+SD of HEK293T transfections performed in triplicates.
  • FIG.15 depicts conformational changes in HSV glycoprotein B (gB).
  • FIG.17 depicts conservation scores determined for amino acids located at positions along an UL27 consensus sequence.
  • the UL27 open reading frame encodes HSV gB.
  • complete HSV-1 and HSV-2 genomes were downloaded from VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2 respectively.
  • FIGS.18A-18D depict four HSV T-cell string polypeptide constructs, referred to as A) RNA construct 1 (Het 1), B) RNA construct 3 (Het 3), C) RNA construct 5 (Het 5), and D) RNA construct 7 (Het 7).
  • RNA construct 1 (Het 1) includes RL2, RL2, RS1 and UL54 T cell antigenic fragments.
  • RNA construct 3 (Het 3) includes UL29, UL39, UL49, and UL9 T cell antigenic fragments.
  • RNA construct 5 (Het 5) includes UL30, UL40, UL5, and UL52 T cell antigenic fragments.
  • RNA construct 7 (Het 7) includes UL1, UL19, UL21, UL27, UL46, UL47, UL25 and UL48 T cell antigenic fragments.
  • FIG.20 shows flow diagrams of Part A and Part B of Example 34. The flow diagrams show an overview of the protocol for safety and immunigenicity of an HSV RNA-based composition as three IM administrations at Visits 1, 4, and 7 in healthy subjects.
  • FIG.21 shows flow diagram of Part C of Example 34.
  • the flow diagram shows an overview of the protocol for safety and immunogenicity of an HSV RNA-based composition as two IM administrations in subjects with a history of genital herpes.
  • Syringe icons represent composition administration.
  • Swab icons represent 28 day twice daily anogenital swabbing periods with daily symptom diary.
  • HSV Herpes simplex virus
  • IM intramuscular
  • Wk week.
  • FIG.22 shows a dose escalating schema for Part A.
  • FIG.23 depicts four HSV antigen constructs referred to as A) RNA construct 1 (Het 1), B) RNA construct 4 (Het 4), C) RNA construct 6 (Het 6), and D) RNA construct 8 (Het 8).
  • RNA construct 1 (Het 1) includes RL2, RL2, RS1 and UL54 T cell antigenic fragments.
  • RNA construct 4 (Het 4) includes UL9, UL49, UL39, and UL29 T cell antigenic fragments.
  • RNA construct 6 (Het 6) includes UL52, UL5.1, UL5.2, UL40, UL30.1, and T cell antigenic fragments.
  • RNA construct 8 (Het 8) includes UL48, UL25, UL47, UL46, UL27.1, UL27.2, UL21, UL19, and UL1 T cell antigenic fragments. HLA-I peptides were detected by mass spectrometry.
  • FIGS.25A-25B depict a graphic overview of all HLA-I epitopes A) and specific A*02:01 epitopes B) in response to RNA construct 4 (Het 4) (SEQ ID NO: 622).
  • FIGS.26A-26B depict a graphic overview of all HLA-I epitopes A) and specific A*02:01 epitopes B) in response to RNA construct 6 (Het 6) (SEQ ID NO: 624).
  • FIGS.27A-27B depict a graphic overview of all HLA-I epitopes A) and specific A*02:01 epitopes B) in response to RNA construct 8 (Het 8) (SEQ ID NO: 626).
  • FIGS.28A-28B depict antigen specific T cell response in mice.
  • A) data is compared to vehicle control;
  • B) data is compared to DMSO. Mice were immunized with 1 ug of each of RNA construct 1 (Het 1), RNA construct 4 (Het 4), RNA construct 6 (Het 6), and RNA construct 8 (Het 8) (Group 5 in Table 23 Example 37).
  • FIGS.29A-29B depict antigen specific T cell response in mice.
  • A) data is compared to vehicle control
  • B) data is compared to DMSO. Mice were immunized with 1 ug of each of RNA construct 1 (Het 1) and RNA construct 8 (Het 8) (Group 6 in Table 23 Example 37).
  • FIGS.30A-30B depict antigen specific T cell response in mice.
  • A) data is compared to vehicle control;
  • B) data is compared to DMSO. Mice were immunized with 1 ug of each of RNA construct 1 (Het 1), RNA construct 4 (Het 4), and RNA construct 6 (Het 6) (Group 7 in Table 23 Example 37).
  • FIG.31 depicts antigen specific T cell response in mice. Mice received saline and data is compared to DMSO.
  • FIG.32 depicts antigen specific T cell response for each T-cell antigenic fragment. Fragments were classified as having a low curated score if the fragments resulted in ⁇ 100 spots per 1x10 6 cells.
  • FIGS.33A-33B depict T-cell response to the antigenic fragments.
  • FIGS.34A-34B depict T-cell response to the antigenic fragments.
  • FIGS.36A-36B depict A) polyfunctional CD4 T- cell response and B) polyfunctional CD8 T cell response in mice immunized with 1 ug of each of RNA construct 1 (Het 1), RNA construct 4 (Het 4), RNA construct 6 (Het 6), and RNA construct 8 (Het 8) (Group 5; total 4 ug, 1 ug of each RNA construct).
  • FIGS.37A-37D depict A) UL54, B) UL29, C) UL40, and D) UL47-specific polyfunctional CD8 T- cell response.
  • FIG.38 shows an exemplary study design.
  • FIG.39 depicts female A02 mice bodyweight 0-12 days post intravaginally challenge with HSV-2 strain MS (5 ⁇ 10 5 PFU (25 LD 50 )).
  • FIG.40 depicts survival curve for female A02 mice 0-12 days post intravaginally challenge with HSV-2 strain MS (5 ⁇ 10 5 PFU (25 LD 50 )).
  • FIGS.41A-41C depict survival curve of female A02 mice immunized with individual RNA constructs. Mice received A) RNA construct 1 (Het 1) or BNT163; B) RNA construct 4 (Het 4) or RNA construct 6 (Het 6); or C) RNA construct 8 (Het 8. BNT163 was used a control: BNT163 is a trivalent composition comprising RNA encoding a glycoprotein C (gC) antigenic fragment, a glycoprotein D (gD) antigenic fragment, and a glycoprotein E (gE) antigenic fragment.
  • FIGS.42A-42C depict survival curve of female A02 mice immunized with a combination of RNA constructs.
  • FIGS.43A-43B depict survival curves.
  • FIGS.44A-44B depict A) survival curve using criteria including clinical readouts such as hindleg paralysis and B) survival curve using criteria excluding hindleg paralysis as a clinical readout for female A02 mice 0- 12 days post intravaginally challenge with HSV-2 strain MS (1x10 6 PFU HSV-2 (500 LD 50 )) immunized with all four construct: RNA construct 1 (Het 1), RNA construct 4 (Het 4), RNA construct 6 (Het 6), and RNA construct 8 (Het 8) (total 4 ug, 1 ug of each RNA construct).
  • FIGS.45A-45B depict cumulative survival days for A) individual RNA constructs; RNA construct 1 (Het 1), RNA construct 4 (Het 4), RNA construct 6 (Het 6), or RNA construct 8 (Het 8) and B) combination of constructs.
  • BNT163 was used a control: BNT163 is a trivalent composition comprising RNA encoding a glycoprotein C (gC) antigenic fragment, a glycoprotein D (gD) antigenic fragment, and a glycoprotein E (gE) antigenic fragment.
  • FIGS.46A-46B depict cumulative survival days for A) all construct and combination of constructs and B) constructs and combination of constructs showing best and worst cumulative survival.
  • BNT163 was used a control: BNT163 is a trivalent composition comprising RNA encoding a glycoprotein C (gC) antigenic fragment, a glycoprotein D (gD) antigenic fragment, and a glycoprotein E (gE) antigenic fragment.
  • FIGS.47A-47D depict vaginal titers using A+B) plaque assay or C+D) qPCR at A+C) 2 days after challenge or B+D) 4 days after challenge.
  • FIG.48 depicts ten HSV antigen constructs: RNA construct 15 (Het 15); RNA construct 16 (Het 16); RNA construct 17 (Het 17); RNA construct 18 (Het 18); RNA construct 19 (Het 19); RNA construct 20 (Het 20); RNA construct 21 (Het 21); RNA construct 22 (Het 22); RNA construct 23 (Het 23); RNA construct 24 (Het 4).
  • RNA construct 15 and RNA construct 16 are immediate early and late constructs and include RL2.1, UL54, UL47, UL46 and UL21 T cell antigenic fragments.
  • RNA construct 17 and RNA construct 18 are early constructs and include UL29, UL39, UL9, and UL5.1, UL40, and UL30.1 T cell antigenic fragments.
  • RNA construct 19 and RNA construct 20 are immediate early and early constructs and include UL2.1, UL54, UL9, UL39, and UL5.1 T cell antigenic fragments.
  • RNA construct 21 and RNA construct 22 are late and early constructs and include UL47, UL46, UL21, UL5.2, UL40, UL30.1, and UL29 T cell antigenic fragments.
  • RNA construct 23 and RNA construct 24 include UL2.1, U54, UL5.2., UL40, UL47, and UL46 cell antigenic fragments.
  • FIGS.49A-49E depict protein expression of the RNA constructs shown in FIG.80 by pair with proteasome inhibitor.
  • FIGS.51A-51B depict antigen specific T cell response in A02 mice under A) loose saturation ( ⁇ 220 spots per 1x10 6 cells) and B) under stringent saturation ( ⁇ 100 spots per 1x10 6 cells). Mice were immunized with 2 ug of RNA construct 15 (Het 15) and 2 ug of RNA construct 17 (Het 17) (Group 1 in Table 24 Example 39).
  • FIGS.52A-52B depict antigen specific T cell response in A02 mice under A) loose saturation ( ⁇ 220 spots per 1x10 6 cells) and B) under stringent saturation ( ⁇ 100 spots per 1x10 6 cells). Mice were immunized with 2 ug of RNA construct 20 (Het 20) and 2 ug of RNA construct 22 (Het 22) (Group 2 in Table 24 Example 39).
  • FIGS.53A-53B depict antigen specific T cell response in A02 mice under A) loose saturation ( ⁇ 220 spots per 1x10 6 cells) and B) under stringent saturation ( ⁇ 100 spots per 1x10 6 cells).
  • FIG.54 depicts antigen specific T cell response in Balb/c mice immunized with 2 ug of RNA construct 15 (Het 15) and 2 ug of RNA construct 17 (Het 17) (Group 1 in Table 24 Example 39).
  • FIG.55 depicts antigen specific T cell response in Balb/c mice immunized with 2 ug of RNA construct 20 (Het 20) and 2 ug of RNA construct 22 (Het 22) (Group 2 in Table 24 Example 39).
  • FIG.56 depicts antigen specific T cell response in Balb/c mice immunized with 4 ug of RNA construct 23 (Het 23) (Group 3 in Table 24 Example 39).
  • FIGS.57A-57C depict CD4 and CD8 T-cell responses in mice immunized with 2 ug of RNA construct 15 (Het 15) and 2 ug of RNA construct 17 (Het 17) (Group 1 in Table 24 Example 39).
  • FIGS.58A-58B depict CD4 and CD8 T-cell responses in mice immunized with 2 ug of RNA construct 15 (Het 15) and 2 ug of RNA construct 17 (Het 17) (Group 1 in Table 24 Example 39).
  • FIGS.60A-60B depict CD4 and CD8 T-cell response in mice immunized with 4 ug of RNA construct 23 (Het 23) (Group 3 in Table 24 Example 39).
  • FIG.61 Immunized mice (Day 0 and Day 21) were injected with medroxyprogesterone (subcutaneous injection 2mg/mouse at Day 46) and challenged intravaginally with HSV-2 strain MS (5x10 3 PFU HSV- 2 ( ⁇ 10xLD 50 )) at Day 51 and monitored for survival, genital disease scoring, weight and vaginal virus titers at 6-h, days 2, 4 and 7.
  • FIGS.62A-62E depict female mice bodyweight 0-16 days post intravaginally challenge with HSV- 2 strain MS (5x10 3 PFU HSV-2 ( ⁇ 10xLD 50 )). Mice were administered with A) PBS (Group 1 in Table 25 Example 40); B) 4 ug of RNA construct 23 (Het 23) (Group 2 in Table 25 Example 40); C) with 2 ug of RNA construct 15 (Het 15) and 2 ug of RNA construct 17 (Het 17) (Group 3 in Table 25 Example 40); D) a trivalent (BNT163) composition comprising RNA encoding a glycoprotein C (gC) antigenic fragment, a glycoprotein D (gD) antigenic fragment, and a glycoprotein E (gE) antigenic fragment; and E) combined graph.
  • A) PBS Group 1 in Table 25 Example 40
  • B) 4 ug of RNA construct 23 (Het 23) Group 2 in Table 25 Example 40
  • FIG.63 depicts survival for female mice 0-15 days post intravaginally challenge with HSV-2 strain MS (5x10 3 PFU HSV-2 ( ⁇ 10xLD 50 )).
  • FIGS.64A-64B depict effect of RNA construct immunization on vaginal disease following HSV-2 infection.
  • FIG.65 depicts vaginal HSV-2 replication kinetics over 7 days.
  • FIG.66 depicts cumulative survival for female mice 0-15 days post intravaginally challenge with HSV-2 strain MS (5x10 3 PFU HSV-2 ( ⁇ 10xLD 50 )).
  • FIGS.67A-67B depict the effect of RNA construct immunizations on HSV-2 replication in mice vaginal cavity.
  • FIGS.68A-68B depict the effect of RNA construct immunizations on HSV-2 replication in mice vaginal cavity. A) day 6-h post challenge; and B) day 7 post challenge.
  • FIG.69 depicts female mice bodyweight 0-16 days post intravaginally challenge with HSV-2 strain MS (5x10 3 PFU HSV-2 ( ⁇ 10xLD 50 )).
  • BNT163 a trivalent composition comprising RNA encoding a glycoprotein C (gC) antigenic fragment, a glycoprotein D (gD) antigenic fragment, and a glycoprotein E (gE) antigenic fragment, .
  • TCS-23 RNA construct 23 (Het 23);
  • TCS 15+17 RNA constructs 15+17 (Het 15+ 17).
  • FIG.70 depicts survival for female mice 0-15 days post intravaginally challenge with HSV-2 strain MS (5x10 3 PFU HSV-2 ( ⁇ 10xLD 50 )).
  • FIGS.71A-71B depict effect of RNA construct immunization on vaginal disease following HSV-2 infection.
  • FIGS.72A-72D depict the effect of RNA construct immunizations on HSV-2 replication kinetics in mice vaginal cavity.
  • BNT163 a trivalent composition comprising RNA encoding a glycoprotein C (gC) antigenic fragment, a glycoprotein D (gD) antigenic fragment, and a glycoprotein E (gE) antigenic fragment
  • FIGS.73A-73F depict expression levels in HEK293T cells transfected with RNA encoding HSV-2 gC (gC2), gD (gD2) and gE (gE2) antigens.
  • FIGS.74A-74F depict expression levels in HEK293T cells transfected with RNA encoding HSV-2 gC (gC2) antigens.
  • FIGS.75A-75B depict expression levels in HEK293T cells transfected with RNA encoding HSV-2 gD (gD2) antigens.
  • FIGS.76A-76D depict expression levels in HEK293T cells transfected with RNA encoding HSV-2 gE (gE2) antigens.
  • FIGS.77A-77C depict secretion levels of HEK293T cells transfected with RNA encoding HSV-2 encoding HSV-2 gC (gC2), gD (gD2) and gE (gE2) antigens.
  • FIG.78 shows cumulative recurrent genital lesion days per group.
  • FIG.79 shows days with recurrent genital lesions for each animal starting 1 day after the second immunization. The same animals as in FIG.78 are shown here.
  • FIG.80 shows cytokine production by CD4+ T cell in response to compositions comprising HSV-2 glycoproteins.
  • Mice were immunized twice as described herein with 10 ⁇ g of an exemplary immunogenic fragment of HSV-2 gB (gB2) RNA-LNP. Splenocytes from these mice were stimulated with a gB2 overlapping peptide pool. CD4+ cytokine-producing T cells were analyzed by flow cytometry.
  • FIG.81 shows cytokine production by CD8+ T cell in response to compositions comprising HSV-2 glycoproteins.
  • FIG.82 depicts expression levels in HEK293T cells transfected with RNA encoding HSV-2 gB (gB2) antigens.
  • FIGS.83A-83B show recurrent genital lesion days per group.
  • Guinea pigs were immunized twice on days 25 and 65 post-infection with nucleoside modified RNAs encapsulated in a lipid nanoparticle and expressing an exemplary HSV-2 gE (gE2) immunogenic fragment and an exemplary HSV-2 gI (gI2) immunogenic fragment (15ug each), or PBS (control). Animals were scored daily Monday to Friday for recurrent genital lesions from 1 day after the first immunization until the end of the study on day 116. From the time of the second immunization, significant differences appeared comparing guinea pigs immunized with modified RNAs encoding exemplary gE2 immunogenic fragment and an exemplary gI2 immunogenic fragment, with the PBS group.
  • FIG.83A shows the cumulative recurrent genital lesion days per group.
  • FIG.83B shows days with recurrent genital lesions for each animal starting 1 day after the second immunization. P values were calculated by the two-tailed Mann Whitney test and demonstrate highly significant differences comparing gE2/gI2 with PBS (**, P ⁇ 0.01; ns, P value not significant).
  • FIGS.84A-84D show T cell responses to gE2 stimulation in mice immunized with gE2/gI2 bivalent RNA composition in CD4+ T cells (FIG.84A) and in CD8+ T cells (FIG.84B); and T cell responses to gI2 stimulation in mice immunized with E2/gI2 bivalent RNA composition in CD4+ T cells (FIG.84C) and in CD8+ T cells (FIG.84D).
  • FIG.85 shows antibody responses to gE2/gI2 in mice immunized with gE2 RNA composition.
  • FIGS.86A-86C show antibody responses to gE2/gI2 (FIG.86A), gE2 (FIG.86B), and gI2 (FIG. 86C) in mice immunized with gE2/gI2 bivalent RNA composition.
  • FIG.87 shows a scheme of the protocol of the mice model experiments described in Example 51
  • FIGS.88A-88C show survival (FIG.88A), weight loss (FIG.88B), and disease score (FIG.88C) in a mouse model of HSV-2 in mice immunized with gE2/gI2 bivalent RNA
  • FIGS.89A-89C show HSV virus titer 2 days (FIG.89A) and 4 days (FIG.89B) after infection in mice immunized with gE2/gI2 bivalent; as well as HSV-2 DNA copy number in DRG 28 days after infection (FIG. 89C).
  • FIGS.91A-91D show the prophylactic effect of BNT163, gB, gE2/gI2, or a combination of them on survival (FIG.91A), disease severity (FIG.91B), genital lesions (FIG.91C), and urinary retention (FIG.91D) in guinea pigs infected with HSV-2.
  • FIGS.92A-92B show the therapeutic effect of BNT163, gB and gE2/gI2 on recurrent lesions in a first (FIG.92A) and the combined results of two experiments (FIG.92B) experiments.
  • the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.
  • Agent may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and/or effect.
  • an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.
  • the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.
  • amino acid refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
  • an amino acid has the general structure H 2 N–C(H)(R)–COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.
  • Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
  • such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
  • such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
  • Antibody agent refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR.
  • CDR complementarity determining region
  • an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
  • an antibody agent may be or comprise a polyclonal antibody preparation.
  • an antibody agent may be or comprise a monoclonal antibody preparation.
  • an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human.
  • an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc.
  • an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains).
  • an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs TM” ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.)).
  • Antigen refers to a molecule that is recognized by the immune system, e.g., in some embodiments the adaptive immune system, such that it elicits an antigen-specific immune response.
  • an antigen-specific immune response may be or comprise generation of antibodies and/or antigen-specific T cells.
  • an antigen is a peptide or polypeptide that comprises at least one epitope against which an immune response can be generated.
  • an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages.
  • an antigen or a processed product thereof such as a T-cell epitope is bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a processed product thereof may react specifically with antibodies or T lymphocytes (T cells).
  • an antigen is a parasitic antigen.
  • an antigen may be delivered by RNA molecules as described herein.
  • a peptide or polypeptide antigen can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length.
  • a peptide or polypeptide antigen can be greater than 50 amino acids. In some embodiments, a peptide or polypeptide antigen can be greater than 100 amino acids.
  • an antigen is recognized by an immune effector cell. In some embodiments, an antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the antigen. In the context of the embodiments of the present disclosure, in some embodiments, an antigen can be presented or present on the surface of a cell, e.g., an antigen presenting cell.
  • an antigen is presented by a diseased cell such as a virus-infected cell.
  • an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC.
  • binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells.
  • binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.
  • Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
  • a particular entity e.g., poypeptide, genetic signature, metabolite, microbe, etc.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • binding typically refers to a non-covalent association between or among entities or moieties. In some embodiments, binding data are expressed in terms of “IC50”.
  • IC50 is the concentration of an assessed agent in a binding assay at which 50% inhibition of binding of reference agent known to bind the relevant binding partner is observed.
  • assays are run under conditions in which the assays are run (e.g., limiting binding target and reference concentrations), these values approximate K D values.
  • Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94/20127 and WO 94/03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol.
  • binding can be expressed relative to binding by a reference standard peptide.
  • a reference standard peptide For example, can be based on its IC 50 , relative to the IC 50 of a reference standard peptide.
  • Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol.2:443 (1990); Hill et al., J. Immunol.147:189 (1991); del Guercio et al., J. Immunol.
  • Cap refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate).
  • a cap is or comprises a guanine nucleotide.
  • a cap is or comprises a naturally-occurring RNA 5’ cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as “m7G.”
  • a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti-reverse cap analogs (ARCAs) known in the art).
  • ARCAs anti-reverse cap analogs
  • a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system).
  • a capped RNA can be obtained by in vitro transcription (IVT) of a single- stranded DNA template in the presence of a dinucleotide or trinucleotide cap analog.
  • Cell-mediated immunity “Cell-mediated immunity,” “cellular immunity,” “cellular immune response,” or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen, in particular characterized by presentation of an antigen with class I or class II MHC.
  • a cellular response relates to immune effector cells, in particular to T cells or T lymphocytes which act as either “helpers” or “killers.”
  • the helper T cells also termed CD4 + T cells or CD4 T cells
  • the killer cells also termed cytotoxic T cells, cytolytic T cells, CD8 + T cells, CD8 T cells, or CTLs
  • kill diseased cells such as virus-infected cells, preventing the production of more diseased cells.
  • co-administration refers to use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent.
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order).
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • an additional therapeutic agent may be combined in one pharmaceutically- acceptable carrier, or they may be placed in separate carriers and delivered to a target cell or administered to a subject at different times.
  • Codon-optimized refers to alteration of codons in a coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule.
  • coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein.
  • codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.”
  • codon- optimization may include increasing guanosine/cytosine (G/C) content of a coding region of RNA described herein as compared to the G/C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.
  • G/C guanosine/cytosine
  • Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
  • the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
  • “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • corresponding to may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition).
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • corresponding to a residue in an appropriate reference polymer.
  • residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190 th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids.
  • sequence alignment strategies including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
  • software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, Scala
  • corresponding to may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity).
  • a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.
  • amino acid sequence “derived from” a designated amino acid sequence (peptide or polypeptide) “derived from” a designated amino acid sequence (peptide or polypeptide), it refers to a structural analogue of a designated amino acid sequence.
  • an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof.
  • Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
  • the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
  • the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.
  • Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
  • individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • Encode refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids.
  • a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme).
  • An RNA molecule can encode a polypeptide (e.g., by a translation process).
  • a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system.
  • a coding region of an RNA molecule encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the RNA sequence of such a target antigen.
  • a coding region of an RNA molecule encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.
  • Engineered refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • Epitope refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
  • an epitope may be recognized by a T cell, a B cell, or an antibody.
  • an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
  • such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation.
  • such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation.
  • an epitope of an antigen may include a continuous or discontinuous portion of the antigen.
  • an epitope is or comprises a T cell epitope.
  • an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.
  • a gene product can be a transcript.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Five prime untranslated region refers to a sequence of an RNA molecule between a transcription start site and a start codon of a coding region of an RNA.
  • “5’ UTR” refers to a sequence of an RNA molecule that begins at a transcription start site and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of an RNA molecule, e.g., in its natural context.
  • fragment as used herein in the context of a nucleic acid sequence (e.g., RNA sequence) or an amino acid sequence may typically be a portion of a reference sequence.
  • a reference sequence is a full-length sequence of e.g., a nucleic acid sequence or an amino acid sequence.
  • a fragment typically, refers to a sequence that is identical to a corresponding stretch within a reference sequence.
  • a fragment comprises a continuous stretch of nucleotides or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total length of a reference sequence from which the fragment is derived.
  • fragment with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus.
  • a fragment of an amino acid sequence comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.
  • homology refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains.
  • Humoral immunity As used herein, the term “humoral immunity” or “humoral immune response” refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
  • Identity refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.
  • Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • an assessed value achieved with a provided pharmaceutical composition may be “increased” relative to that obtained with a comparable reference pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine).
  • an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein.).
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.
  • the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference.
  • the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.
  • the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.
  • Ionizable refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH.
  • an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values.
  • an ionizable amino lipid may have a pKa within a range of about 5 to about 7.
  • Isolated means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”.
  • RNA lipid nanoparticle As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s).
  • an RNA lipid nanoparticle comprises at least one ionizable amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm.
  • an average size e.g., Z-average
  • RNA lipid nanoparticles can have a particle size (e.g., Z- average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm.
  • a particle size e.g., Z- average
  • an average size of lipid nanoparticles is determined by measuring the particle diameter.
  • RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.
  • Lipidoid As used herein, a “lipidoid” refers to a lipid-like molecule. In some embodiments, a lipoid is an amphiphilic molecule with one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoids.
  • Nanoparticle As used herein, the term “nanoparticle” refers to a particle having an average size suitable for parenteral administration.
  • a nanoparticle has a longest dimension (e.g., a diameter) of less than 1,000 nanometers (nm). In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm. In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 ⁇ m and about 500 nm, or between about 1 nm and 1,000 nm.
  • a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm.
  • a nanoparticle may be substantially spherical so that its longest dimension may be its diameter.
  • a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. [0137]
  • Naturally occurring The term “naturally occurring” as used herein refers to an entity that can be found in nature.
  • Neutralization refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.
  • Nucleic acid particle can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a nucleic acid particle may comprise at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid.
  • a nucleic acid particle is a lipid nanoparticle.
  • a nucleic acid particle is a lipoplex particle.
  • nucleic acid refers to a polymer of at least 10 nucleotides or more.
  • a nucleic acid is or comprises DNA.
  • a nucleic acid is or comprises RNA.
  • a nucleic acid is or comprises peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a nucleic acid is or comprises a single stranded nucleic acid.
  • a nucleic acid is or comprises a double-stranded nucleic acid.
  • a nucleic acid comprises both single and double-stranded portions.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues.
  • natural residues e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil.
  • a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).
  • a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.
  • Nucleotide refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.
  • Patient refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non- human primates, and/or humans).
  • a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes an HSV infection. In some embodiments, a patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. In some embodiments, a patient is a patient suffering from or susceptible to an HSV infection.
  • PEG-conjugated lipid refers to a molecule comprising a lipid portion and a polyethylene glycol portion.
  • Pharmaceutical composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection as, for example, a sterile solution or suspension formulation.
  • compositions comprising: pharmaceutically effective amount or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • a desired reaction in some embodiments relates to inhibition of the course of the disease. In some embodiments, such inhibition may comprise slowing down the progress of a disease and/or interrupting or reversing the progress of the disease.
  • a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease or a condition.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • an effective amount of pharmaceutical compositions will depend, for example, on a disease or condition to be treated, the severity of such a disease or condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
  • Poly(A) sequence As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical.
  • RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • Polypeptide refers to a polymeric chain of amino acids.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids.
  • a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof.
  • such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
  • a polypeptide may be cyclic, and/or may comprise a cyclic portion.
  • a polypeptide is not cyclic and/or does not comprise any cyclic portion.
  • a polypeptide is linear.
  • a polypeptide may be or comprise a stapled polypeptide.
  • the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a common sequence motif e.g., a characteristic sequence element
  • shares a common activity in some embodiments at a comparable level or within a designated range
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
  • a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • Recombinant in the context of the present disclosure means “made through genetic engineering”. In some embodiments, a “recombinant” entity such as a recombinant nucleic acid in the context of the present disclosure is not naturally occurring.
  • Reference As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
  • RNA Ribonucleic acid
  • RNA Ribonucleic acid
  • polyribonucleotide refers to a polymer of ribonucleotides.
  • an RNA is single stranded.
  • an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above.
  • An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region.
  • an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a RNA to a ribosome to initiate translation.
  • an RNA is a synthetic RNA.
  • Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).
  • Ribonucleotide encompasses unmodified ribonucleotides and modified ribonucleotides.
  • unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U).
  • Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g.
  • ribonucleotide also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.
  • risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • RNA lipoplex particle refers to a complex comprising liposomes, in particular cationic liposomes, and RNA molecules. Without wishing to bound by a particular theory, electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles.
  • positively charged liposomes may comprise a cationic lipid, such as in some embodiments DOTMA, and additional lipids, such as in some embodiments DOPE.
  • an RNA lipoplex particle is a nanoparticle.
  • specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non- specific binding moiety.
  • Subject refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., an HSV infection). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., an HSV infection).
  • a disease, disorder, or condition e.g., an HSV infection
  • a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., an HSV infection). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., an HSV infection). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., an HSV infection). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., an HSV infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Suffering from An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.
  • Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public.
  • an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition. [0159] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring.
  • a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis.
  • the term “synthetic” refers to an entity that is made outside of biological cells.
  • a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.
  • a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • a medical intervention e.g., surgery, radiation, phototherapy
  • 3' UTR refer to a sequence of an RNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context.
  • Threshold level refers to a level that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay.
  • a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g., a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria).
  • a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population.
  • a threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.
  • Treat As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition.
  • Vaccination refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent.
  • vaccination can be administered before, during, and/or after exposure to a disease-associated agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent.
  • vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition.
  • vaccination generates an immune response to an infectious agent.
  • Vaccine refers to a composition that induces an immune response upon administration to a subject. In some embodiments, an induced immune response provides protective immunity.
  • Variant As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • moieties e.g., carbohydrates, lipids, phosphate groups
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
  • a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference.
  • a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference.
  • a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference.
  • a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference.
  • a reference polypeptide or nucleic acid is one found in nature.
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • vectors are referred to herein as “expression vectors.”
  • known techniques may be used, for example, for generation or manipulation of recombinant DNA, for oligonucleotide synthesis, and for tissue culture and transformation (e.g., electroporation, lipofection).
  • Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • HSV herpes simplex virus
  • HSV-2 antigen constructs e.g., HSV-2 antigen constructs, or a combination thereof
  • HSV herpes simplex virus
  • the present disclosure provides HSV (e.g., HSV-1, HSV-2, or both) compositions (e.g., immunogenic compositions, e.g., vaccines) and related technologies (e.g., methods).
  • the present disclosure provides for example, polyribonucleotides that encode one or more GP polypeptides that each comprise an HSV glycoprotein or an antigenic portion thereof.
  • the present disclosure also provides, for example, polyribonucleotides that encode one or more T-cell string polypeptides that each comprises one or more HSV T-cell antigens or antigenic portions threof.
  • a polyribonucleotide provided herein can be part of an RNA construct.
  • a polyribonucleotide or RNA construct as described herein can be part of a composition (e.g., a pharmaceutical composition, e.g., an immunogenic composition, e.g., a vaccine).
  • HSV Herpes Simplex Virus
  • HSV-1 and HSV-2 The structure of HSV-1 and HSV-2 mainly include (from inside to outside) a DNA core, capsid, tegument and envelope. Each of HSV-1 and HSV-2 have a double stranded DNA genome of about 153kb, encoding at least 80 genes.
  • the DNA core is enclosed by an icosapentahedral capsid composed of 162 capsomeres, 150 hexons and 12 pentons, made of six different viral proteins.
  • the DNA is surrounded by at least 20 different viral tegument proteins that have structural and regulatory roles. Some of them participating in capsid transport to the nucleus and other organelles, viral DNA entry into the nucleus, activation of early genes transcription, suppression of cellular protein biosynthesis, and RNA degradation.
  • the viral envelope surrounding the tegument has at least 12 different glycoproteins (B-N) on their surface.
  • the glycoproteins may exist as heterodimers (H/L and E/I) with most existing as monomers.
  • HSV-1 and HSV-2 are responsible for a number of minor, moderate and severe pathologies, including oral and genital ulceration, virally induced blindness, viral encephalitis and disseminated infection of neonates. HSV-1 and HSV-2 are usually transmitted by different routes and affect different areas of the body, but the signs and symptoms that they cause can overlap. Infections caused by HSV-1 represent one of the more widespread infections of the orofacial region and commonly causes herpes labialis, herpetic stomatitis, and keratitis. HSV-2 typically causes genital herpes and is transmitted primarily by direct sexual contact with lesions.
  • HSV infections are transmitted through contact with herpetic lesions, mucosal surfaces, genital secretions, or oral secretions. The average incubation period after exposure is typically 4 days, but may range between 2 and 12 days. HSV particles can infect neuronal prolongations enervating peripheral tissues and establish latency in these cells, namely in the trigeminal ganglia and dorsal root ganglia of the sacral area from where they can sporadically reactivate.
  • HSV infections are lifelong and generally asymptomatic. Without wishing to be bound by any particular theory, it is understood that HSV particles can be shed from infected individuals independent of the occurrence of clinical manifestations. [0175] HSV infections are rarely fatal, but are characterized by blisters that can rupture and become painful. There are few clear differences in clinical presentation based on the type of infecting virus. However, as discussed above, HSV-1 infections tend to be less severe than HSV-2 infections, and patients infected with HSV-2 generally have more outbreaks. A.
  • an HSV (HSV-1 or HSV-2) particle binds to the cell surface using the viral glycoproteins and fuses its envelope with the plasma membrane (see, e.g., FIG.2, Step 1).
  • the viral capsid and tegument proteins are internalized in the cytoplasm (see, e.g., FIG.2, Step 2).
  • the viral capsid Once in the cytoplasm, the viral capsid accumulates in the nucleus and releases viral DNA into the nucleus (see, e.g., FIG.2, Step 3).
  • HSV replicates by three rounds of transcription that yield: ⁇ (immediate early) proteins that mainly regulate viral replication; ⁇ (early) proteins that synthesize and package DNA; and ⁇ (late) proteins, most of which are virion proteins (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar 1;7:d752-64; and Ibá ⁇ ez et.al., Front Microbiol. 2018 Oct 11;9:2406; each of which is incorporated herein by reference in its entirety) (see, e.g., FIG.2, Steps 4-6).
  • HSV capsids are assembled within the nucleus of infected cells (see, e.g., FIG..2, Step 7). Once the assembly of viral capsids has been completed in the nucleus, these particles will continue their maturation process in this same compartment through the acquisition of tegument proteins. After leaving the nucleus, additional tegument proteins will be added to the capsids. Meanwhile, the glycoproteins are translated and glycosylated in the endoplasmic reticulum and processed in the trans-Golgi network (TGN) and then directed to multivesicular bodies (see, e.g., FIG.2, Step 8).
  • TGN trans-Golgi network
  • HSV HSV-1 or HSV-2
  • HSV are able to establish a latent infection. After primary infection, HSV either replicates productively in epithelial cells or enters sensory neuron axons and moves to the neuronal cell nucleus.
  • the viral DNA remains as circular, extra-chromosomal DNA, and does not possess any lytic gene expression; however, latency associated transcripts are expressed and then spliced to produce RNA.
  • This general transcriptional silence may allow the virus to remain hidden in the cell by avoiding immune surveillance.
  • technologies e.g., compositions and methods for augmenting, inducing, promoting, enhancing and/or improving an immune response against HSV (e.g., HSV-1 and/or HSV-2) or a component thereof (e.g., a protein or portion thereof).
  • technologies provided herein are designed to augment, induce, promote, enhance and/or improve immunological memory against HSV or a component thereof (e.g., a protein or portion thereof).
  • technologies described herein are designed to act as an immunological boost to a primary composition (e.g., immunogenic composition, e.g., vaccine), such as a composition (e.g., immunogenic composition, e.g., vaccine) directed to an epitope and/or epitopes of HSV (e.g., HSV-1 and/or HSV-2).
  • HSV Genome The genome of HSV-1 and the genome of HSV-2 are both approximately 150 kb long of double- stranded DNA, varying slightly between subtypes and strains. The genome encodes more than 80 genes and has high GC contents: 67 and 69% for HSV-1 and HSV-2, respectively (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar 1;7:d752-64; and Jiao et.al., Microbiol Resour Announc.
  • the genome is organized as unique long region (UL) and a unique short region (US).
  • the UL is typically bounded by terminal long (TRL) and internal long (IRL) repeats.
  • the US is typically bounded by terminal short (IRS) and internal short (TRS) repeats.
  • TRL terminal long
  • INL internal long
  • TRS terminal short
  • TRS internal short
  • HSV contains three origins of replication within the genome that are named depending upon their location in either the Long (oriL) or Short (oriS) region of the genome. OriL is found as a single copy in the UL segment, but oriS is located in the repeat region of the Short segment; thus, it is present in the genome in two copies. Both oriL and oriS are palindromic sequences consisting of an AT-rich center region flanked by inverted repeats that contain multiple binding sites of varying affinity for the viral origin binding protein (UL9).
  • UL9 viral origin binding protein
  • oriL or one of the oriS sequences is sufficient for viral replication (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar 1;7:d752-64; and Jiao et.al., Microbiol Resour Announc. 2019 Sep; 8(39): e00993-19, which is incorporated herein by reference in its entirety).
  • the viral genome also contains signals that orchestrate proper processing of the newly synthesized genomes for packaging into pre-formed capsids. Progeny genomes are generated in long concatemers that require cleavage into unit-length monomers.
  • the viral genome contains two DNA sequence elements, pac1 and pac2, that ensure proper cleavage and packaging of unit-length progeny genomes. These elements are located within the direct repeats (DR) found within the inverted repeat regions at the ends of the viral genome (see, Whitley et.al., Lancet 2001 May 12;357(9267); Taylor et.al., Front Biosci. 2002 Mar 1;7:d752-64; and Jiao et.al., Microbiol Resour Announc. 2019 Sep; 8(39): e00993-19, which is incorporated herein by reference in its entirety).
  • DR direct repeats
  • ICP0 Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an ⁇ (immediate-early) protein of herpes simplex virus 1, and is capable of activating HSV-1 gene expression, disrupt nuclear domain (ND) 10 structures, mediate the degradation of cellular proteins, and evade the host cell’s intrinsic and innate antiviral defenses (see., Smith et.al., Future Virol. 2011 Apr; 6(4): 421–429).
  • ICP22 ICP22
  • ICP22 is expressed from an immediate–early (IE) gene during the replication cycle of HSV-1 and HSV-2.
  • ICP22 can generally regulate viral and host gene transcription by changing the phosphorylation status of host RNA polymerase II (RNA pol II) and can also facilitate the nuclear egress complex (NEC) accurately locate to the nuclear membrane to promote nuclear budding (see, Wu et.al., Front Microbiol. 2021 Jun 7;12:668461).
  • RNA pol II RNA polymerase II
  • NEC nuclear egress complex
  • the UL48 gene encodes VP16 or alpha-gene-transactivating factor ( ⁇ -TIF).
  • VP16 is an important transactivator that can activate the transcription of viral immediate-early genes, and in the late stage of viral replication. Additionally, VP16, as a tegument, is involved in viral assembly (see Fan, et.al., Front Microbiol.
  • VP16 released by invading virions binds to the immediate-early (IE) gene promoter to stimulate the transcription of IE genes as a transactivating factor that acts specifically on IE genes (see Fan, et.al., Front Microbiol. 2020; 11: 1910).
  • IE immediate-early
  • VP16 assembles into the tegument to participate in the assembly of virions and promote their maturation (see Fan, et.al., Front Microbiol. 2020; 11: 1910).
  • Glycoproteins [0188] In order to replicate, enveloped HSV must be able to fuse with the membrane of a living cell and deliver their genetic material into its cytoplasm.
  • the HSV viral envelope surrounding the tegument has at least 12 different glycoproteins (gB-gN) on their surface.
  • the glycoproteins may exist as heterodimers (gH/gL and gE/gI) with most existing as monomers.
  • HSV gC, gB, gD, gH, and gL are involved in the process of viral cell entry. Initial attachment is mediated by gC, followed by gD. Then gH/gL pull the virus and the cell membrane together, and then gB triggers the membrane fusion. (Reske et. al., Rev Med Virol. May-Jun 2007; and Arii et. al., Adv Exp Med Biol. 2018;1045:3-21).
  • HSV glycoprotein e.g., gB, gC, gD, gE, gG, gH, gI, and/or gL
  • HSV glycoprotein e.g., gB, gC, gD, gE, gG, gH, gI, and/or gL
  • antigens and antigenic fragments thereof can be useful in preventing or treating HSV, e.g., in HSV antigen constructs and/or HSV compositions (e.g., immunogenic compositions, e.g., vaccines) as further disclosed herein.
  • HSV glycoprotein e.g., gB, gC, gD, gE, gG, gH, gI, and/or gL
  • HSV glycoprotein e.gB, gC, gD, gE, gG, gH, gI, and/or gL
  • Glycoprotein C is a type I membrane glycoprotein and is considered a significant attachment protein and principle viral ligand for binding heparin sulfate proteoglycans (HSPGs) on a cell surface. This binding can occur by gC interaction with HSPG rich regions found on F-actin rich membrane protrusions referred to as filopodia. [0191] Glycoprotein C has also been shown to be involved in regulation of cell entry and infection by increasing pH threshold for acid-induced conformational changes of gB. Low pH induces reversible conformational changes to gB domains I and V, the functional region containing hydrophobic loops important in cell fusion.
  • HSPGs heparin sulfate proteoglycans
  • Glycoprotein C By positively regulating low-pH-induced conformational changes of gB, gC can enhance HSV’s ability to invade cell types, like epithelial cells, that require a low-pH mechanism for invasion. [0192] Glycoprotein C has also been shown to play a role in immune evasion, in addition to its role in attachment. Glycoprotein C is a target for lymphocyte cytotoxicity in certain cell types and is able to bind complement component C3b to inhibit compliment activation. Furthermore, neutralizing epitopes that exist on other HSV glycoproteins, like gB, can be protected by gC, preventing immune responses from blocking fusion. 6.
  • HSV glycoprotein D (gD) is a 46 kDA type I membrane glycoprotein.
  • the N-terminal ectodomain is comprised of 316 amino acids.
  • Glycoprotein D facilitates invasion by interacting with several cell surface receptors, including herpesvirus entry mediator (HVEM), nectin-1 or nectin-2, and heparin sulfate that contain specific modifications. These cellular receptors do not function as co-receptors, as each glycoprotein interaction with a cell’s receptor occurs independently of each other.
  • HVEM herpesvirus entry mediator
  • nectin-1 or nectin-2 heparin sulfate that contain specific modifications.
  • HVEM the first gD receptor identified, belongs to the tumor necrosis factor receptor family and is commonly found on T cells, B cells, dendritic cells, natural killer cells, macrophages, as well as non-immune cell types like neurons and epithelial cells. Within the N-terminus of gD, there is a 37 residue hairpin structure that forms the entire site for binding to HVEM.
  • HVEM cysteine-rich domain 1.
  • this N-terminal extension adopts an extended and flexible conformation.
  • Clinical strains of HSV use nectin-1 for cell entry; however, several mutant strains of HSV utilize nectin-2. Furthermore, heparin sulfate is utilized by HSV-1 but not HSV-2. Glycoprotein D interaction with net-1 has been shown to be essential in some cell types such as neurons, even when other receptors are present on a cell surface. 7.
  • Glycoprotein H (gH)/Glycoprotein L (gL) Complex [0195] Glycoprotein H (gH) is an essential 56kD protein that exists as a heterodimeric complex with 25 kDa glycoprotein L (gL) (complex referred to herein as gH/gL). The gH/gL complex is required for cell fusion and entry. gH/gL does not share any structural similarities with documented fusion proteins and likely does not function as a cofusogen with gB. Instead, gH/gL may act as a regulator of fusion and important component in stabilizing contact between HSV and a cell.
  • Glycoprotein H receives a signal from gD through its H1 domain, and transmits this signal to membrane proximal H3 domain, which in turn propagates that signal to gH’s cytoplasmic tail.
  • gH’s cytoplasmic tail Once gH’s cytoplasmic tail receives this signal, it releases strain on the pre-fusion conformation of gB, which favors attachment of gB’s fusion loop to a cell surface, promoting gB mediated membrane fusion.
  • Mutations in gH’s C-terminal tail have been shown to reduce fusion activity.
  • antibody responses directed towards gH have been shown capable of inhibiting fusion processes mediated by gB-gH-gL.
  • Glycoprotein B is a protein that has an apparent molecular weight of approximately 95-100 kDa and consists of an extended rod or spike-like ectodomain, a hydrophobic membrane proximal region (MPR), a transmembrane region (TMR), and a C-terminal domain (CTD).
  • Glycoprotein B is a class III fusogen. Glycoprotein B ectodomain architecture shares conformational similarity with fusogens from viruses not belonging to the herpesvirdae family. Glycoprotein B is activated through its interaction with gH/gL, but HSV cannot fuse with a target cell through activation of gB alone and requires gB interaction to specific receptors for fusion to be completed.
  • a well-known receptor target of gB is cell-surface heparin sulfate, an interaction that is not essential for HSV fusion, but is known to promote viral adhesion to a cell surface.
  • HSV gB exists in two forms, a pre-fusion and post fusion form. Several changes in the pre-fusion form of gB are thought to lead to its active and post-fusion state. The first change occurs at domain V or at MPR, which allows fusion loops to point towards a cell membrane and away from a viral membrane. This change can produce a compacting intermediate conformation 1 that does not yet attach to a cell membrane surface. The next change occurs at domain III and involves gB adopting an extended intermediate conformation 2 that allows its fusion loop to attach to a cell membrane surface.
  • domain V converts gB to its post-fusion conformation that favors membrane fusion.
  • the post-fusion form of HSV-1 gB has an ectodomain that exists as three protomers that interact to produce a rod-like trimeric structure. Each promoter is comprised of five distinct domains with linker regions that individually form a hairpin shape. Each domain of an individual protomer interacts with the same domain of an adjacent protomer to form the described trimeric structure.
  • Domain I houses an important fusion loop and is commonly referred to as the fusion domain.
  • Domain II facilitates interactions with gH/gL and is referred to as the gH/gL domain.
  • Domain III is comprised of alpha helices that help form the trimeric coil-coil central core of this protein.
  • Domain IV is referred to as the crown domain and sits on top of the post-fusion form; it is believed to bind with cellular receptors. Antibodies that bind to the crown domain can disrupt gB binding to cellular receptors. Domain V consists of a long extension and connects protomers together. 9. Glycoprotein E and glycoprotein I (gE/gI) [0199] Glycoprotein E is approximately 53 kDa and Glycoprotein I is approximately 141 kDa. Both proteins interact to form a heterodimeric complex (complex referred to herein as gE/gI) that plays a role in cell-to-cell spread and virus induced fusion.
  • gE/gI glycoprotein I
  • the gE/gI complex unlike gB, gD, and gH/gL, is not required for fusion and entrance into a cell, but is important for cell-to-cell spread. Disruption of gE/gI formation has effects on HSV proliferation, as this virus relies on cell-to-cell spread for its lytic cycle.
  • the mechanism in which gE/gI facilitate cell-to-cell spread is thought to be reliant on several tegument polypeptides. Cooperation of tegument polypeptides, UL11, UL16, and UL21 may play a role in processing, transport, and biological activity of gE. 10.
  • Glycoprotein G from both HSV-1 (gG1) and HSV-1 (gG2) is the first viral chemokine-binding protein shown to potentiate chemokine function of a cell.
  • Glycoprotein G varies in size significantly between HSV-1 and HSV-2, with a 76 kDa and 43 kDa size, respectively.
  • Glycoprotein G is unique in that its soluble form (SgG2) can have immune modulatory capacity through its extracellular activity.
  • SgG2 binds chemokines through the glycosaminoglycan (GAG)-binding domain of a chemokine without interfering with chemokine’s G protein coupled receptors (GPCRs) binding site.
  • GAG glycosaminoglycan
  • GPCRs G protein coupled receptors
  • SgG2’s interaction with GAG containing proteins allows initiation of lipid raft formation and accumulation, which produces a clustering of chemokine receptors into this micro domain. Clustering of chemokine receptors, in turn, increases local concentration of chemokines on a host cell’s extracellular surface and allows these chemokines to interact with GPCRs. This interaction likely leads to increased immune signaling responses and chemokine stimulation.
  • ICP47 Infected cell protein 47 (ICP47) encoded by gene US12, is a polymorphous protein and could block RNA splicing in early infection, and then, shuttle viral RNA from nucleus to cytoplasm in late infection.
  • ICP47 directly binds antigen-dependent transporter (TAP), limiting antigen trafficking, leading to the occurrence of empty MHC-I (Cheng et.al., Virol J. 2020 Jul 10;17(1):101).
  • TAP antigen-dependent transporter
  • the binding of ICP47 to TAP stabilizes the inward conformation, therefore blocking the translocation pathway points to the endoplasmic reticulum (ER) cavity.
  • ER endoplasmic reticulum
  • VHS virion-host shutoff
  • the virion-host shutoff (VHS) protein is viral protein synthesized with late kinetics and packaged into mature virion particles. Functionally, VHS is a viral RNase that preferentially degrade both host and viral RNA species. VHS has been reported to interfere with dendritic cells (DC) activation during both productive and nonproductive HSV infection (Cotter et.al., J Virol. 2011 Dec; 85(23): 12662–12672.). 13. US3 [0203] All members of the Alphaherpesvirinae subfamily encode a serine/threonine kinase, designated US3.
  • HSV-1 herpes simplex virus type 1
  • nAbs neutralizing antibodies
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • T cell activity in some embodiments, CD4 T cell activity, in some embodiments CD8 T cell activity, in some embodiments, both.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • CD4 and CD8 epitope(s) of one or more HSV antigens e.g., HSV-1 antigens, HSV-2 antigens, or a combination thereof
  • HSV antigens e.g., HSV-1 antigens, HSV-2 antigens, or a combination thereof
  • B cell antigens and/or epitopes e.g., in addition to one or more B cell antigens and/or epitopes
  • Table 1 Certain HSV Compositions Under Clinical Development Name Platform Antigens Immune Clinical results Response is , is c c c y Failed to reduce shedding c
  • the present disclosure provides the recognition that constructs and/or compositions described herein may be administered as part of regimen with other therapeutic agents.
  • an anti-viral agent can be administered to treat HSV-1 or HSV-2 infection or recurrent episodes.
  • an anti-viral agent is or comprises acyclovir, valacyclovir, famciclovir, or a combination thereof. Table 2 below provides certain information about select anti-viral agents.
  • the present invention provides combinations of nucleotides (e.g., polyribonucleotides) that can be used to express and/or deliver one or more HSV polypeptides or an antigenic portion thereof.
  • nucleotides e.g., polyribonucleotides
  • the present disclosure provides a combination comprising a plurality of polyribonucleotides.
  • the plurality of polyribonucleotides comprise a first set of polyribonucleotides. In some embodiments the plurality of polyribonucleotides comprise a first set of polyribonucleotides and a second set of polyribonucleotides. In some embodiments, a first set of polyribonucleotides encode one or more glycoprotein (GP) polypeptides. In some embodiments, a second set of polyribonucleotides encode one or more T-cell string polypeptides.
  • GP glycoprotein
  • the present disclosure provides the insight that delivering one or more GP polypeptides and one or more T-cell string polypeptides to a subject can induce a more robust immune response to HSV (e.g., HSV-1, HSV-2 or both).
  • HSV e.g., HSV-1, HSV-2 or both
  • GP polypeptides can induce humoral immune responses (e.g., B cell response) to portions of an HSV particle that are surface exposed in a subject prior to infection
  • T-cell string polypeptides can induce cell-mediated immune responses (e.g., T cell response) to HSV molecules (e.g., proteins) produced inside a subject’s cells following infection.
  • a first set of polyribonucleotides can include one or more polyribonucleotides.
  • a polyribonucleotide in a first set can encode a glycoprotein (GP) polypeptide.
  • GP glycoprotein
  • a GP polypeptide comprises an HSV glycoprotein or an antigenic portion thereof.
  • a first set of polyribonucleotides encodes two or more GP polypeptides.
  • a first set of polyribonucleotides comprises two or more polyribonucleotides.
  • two or more GP polypeptides comprise an HSV glycoprotein or an antigenic portion thereof.
  • the two or more GP polypeptides differ.
  • a first set of polyribonucleotides encode three or more GP polypeptides.
  • a first set of polyribonucleotides comprises three or more polyribonucleotides.
  • three or more GP polypeptides comprise an HSV glycoprotein or an antigenic portion thereof. In some embodiments, the three or more GP polypeptides differ.
  • a first set of polyribonucleotides encode four or more GP polypeptides. In some embodiments, a first set of polyribonucleotides comprises four or more polyribonucleotides. In some embodiments, four or more GP polypeptides comprise an HSV glycoprotein or an antigenic portion thereof. In some embodiments, the four or more GP polypeptides differ.
  • a first set of polyribonucleotides encode one or more GP polypeptides, wherein the one or more GP polypeptides comprise an HSV glycoprotein C (gC) or an antigenic portion thereof, an HSV glycoprotein D (gD) or an antigenic portion thereof, an HSV glycoprotein E (gE) or an antigenic portion thereof, an HSV glycoprotein B (gB) or an antigenic portion thereof, an HSV glycoprotein I (gI) or an antigenic portion thereof, an HSV glycoprotein G (gG) or an antigenic portion thereof, an HSV glycoprotein H (gH) or an antigenic portion thereof, an HSV glycoprotein L (gL) or an antigenic portion thereof, or a combination thereof.
  • the one or more GP polypeptides comprise an HSV glycoprotein C (gC) or an antigenic portion thereof, an HSV glycoprotein D (gD) or an antigenic portion thereof, an HSV glycoprotein E (gE) or an antigenic portion thereof, an HSV glycoprotein B (gB) or
  • a first set of polyribonucleotides comprises (i) a polyribonucleotide encoding an HSV gC or an antigenic portion thereof, (ii) a polyribonucleotide encoding an HSV gD or an antigenic portion thereof, (iii) a polyribonucleotide encoding an HSV gE or an antigenic portion thereof, or (iv) a combination thereof.
  • a first set of polyribonucleotides comprises (i) a polyribonucleotide encoding an HSV gB or an antigenic portion thereof, (ii) a polyribonucleotide encoding an HSV gC or an antigenic portion thereof, (iii) a polyribonucleotide encoding an HSV gD or an antigenic portion thereof, or (iv) a combination thereof.
  • a first set of polyribonucleotides comprises (i) a polyribonucleotide encoding an HSV gC or an antigenic portion thereof, (ii) a polyribonucleotide encoding an HSV gD or an antigenic portion thereof, (iii) a polyribonucleotide encoding an HSV gE or an antigenic portion thereof, (iv) a polyribonucleotide encoding an HSV gB or an antigenic portion thereof, or (v) a combination thereof.
  • a first set of polyribonucleotides comprises: (i) a polyribonucleotide that encoding an antigenic portion of HSV gC, (ii) a polyribonucleotide that encoding an antigenic portion of HSV gD, (iii) a polyribonucleotide that encoding an antigenic portion of HSV gE, or (iv) a combination thereof.
  • a first set of polyribonucleotides comprises: (i) a polyribonucleotide that encoding an antigenic portion of HSV gC, (ii) a polyribonucleotide that encoding an antigenic portion of HSV gD, (iii) a polyribonucleotide that encoding an antigenic portion of HSV gE, (iv) a polyribonucleotide that encoding an antigenic portion of HSV gB, or (v) a combination thereof.
  • the first set of polyribonucleotides comprises (i) a polyribonucleotide that encoding an antigenic portion of HSV gC, (ii) a polyribonucleotide that encoding an antigenic portion of HSV gD, and (iii) a polyribonucleotide that encoding an antigenic portion of HSV gE.
  • the first set of polyribonucleotides comprises (i) a polyribonucleotide that encoding an antigenic portion of HSV gC, (ii) a polyribonucleotide that encoding an antigenic portion of HSV gD, (iii) a polyribonucleotide that encoding an antigenic portion of HSV gE, and (iv) a polyribonucleotide that encoding an antigenic portion of HSV gB.
  • the present invention provides one or more glycoprotein (GP) polypeptides each comprising an HSV glycoprotein or an antigenic portion thereof.
  • an HSV glycoprotein or an antigenic portion thereof is an HSV-1 glycoprotein or an antigenic portion thereof. In some embodiments, an HSV glycoprotein or an antigenic portion thereof is an HSV-2 glycoprotein or an antigenic portion thereof. [0225] An overview of exemplary HSV glycoproteins with or without a secretory signal is provided in Table 3 below.
  • Table 3 Exemplary HSV Glycoprotein, Secretory Signal, and Versions HSV-2 Secretory Amino nt Version 1 Version 2 Version 3 Version 4 Glycoprotein signal acid SEQ ID nt nt nt nt SEQ ID NO SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID HSV-2 gE – 327 325 LVVV (SEQ ID NO: Table 4 below, example deoxyribonucleic acid sequences encoding certain HSV gB, gC, gD, and gE glycoproteins are provided in Table 5, and example ribonucleic acid sequences encoding certain HSV gB, gC, gD, and gE glycoproteins are provided in Table 6 below.
  • a first set of polyribonucleotides encode one or more glycoprotein (GP) polypeptides.
  • one or more GP polypeptides each comprise an HSV glycoprotein, a variant thereof, or an antigenic portion thereof.
  • one or more GP polypeptides comprise an HSV glycoprotein, a variant thereof, or an antigenic portion thereof as set forth in Table 4.
  • a GP polypeptide comprises an HSV glycoprotein C (gC) or antigenic portions thereof.
  • a GP polypeptide comprises an antigenic portion of HSV gC.
  • an antigenic portion of HSV gC comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or a portion thereof.
  • an antigenic portion of HSV gC has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1.
  • an antigenic portion of HSV gC comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 260 or a portion thereof.
  • an antigenic portion of HSV gC has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 260.
  • At least one polyribonucleotide of the first set of polyribonucleotides comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ribonucleic acid sequence of any one of SEQ ID NO: 16-19, 147 and 274-281.
  • a GP polypeptide comprises an HSV glycoprotein D (gD) or antigenic portions thereof.
  • a GP polypeptide comprises an antigenic portion of HSV gD.
  • an antigenic portion of HSV gD comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or a portion thereof.
  • an antigenic portion of HSV gD has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 2.
  • At least one polyribonucleotide of the first set of polyribonucleotides comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ribonucleic acid sequence of any one of SEQ ID NO: 20-23, 143, and 286.
  • a GP polypeptide comprises an HSV glycoprotein E (gE) or antigenic portions thereof.
  • a polypeptide comprises an HSV gE antigens or antigenic portions thereof.
  • an antigenic portion of HSV gE comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3 or a portion thereof.
  • an antigenic portion of HSV gE has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 3.
  • At least one polyribonucleotide of the first set of polyribonucleotides comprises a ribonucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ribonucleic acid sequence of any one of SEQ ID NO: 24-27 and 282-285.
  • the present disclosure provides a combination comprising a plurality of polyribonucleotides.
  • a plurality of polyribonucleotides comprise a first set of polyribonucleotides.
  • a first set of polyribonucleotides encode one or more glycoprotein (GP) polypeptides.
  • at least one polyribonucleotide of the polyribonucleotides in the first set encodes a GP polypeptide that comprises an HSV glycoprotein (gB) variant thereof, or one or more antigenic portions thereof.
  • GP glycoprotein
  • At least one polyribonucleotide of the polyribonucleotides in the first set encodes a GP polypeptide that comprises an antigenic portion of HSV glycoprotein (gB) or an antigenic portion of a variant of HSV gB.
  • a GP polypeptide comprises an HSV glycoprotein B (gB), variant thereof, or antigenic portions thereof.
  • a GP polypeptide comprises an antigenic portion of HSV gB.
  • an HSV gB comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 379 or a portion thereof.
  • an antigenic portion of HSV gB comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 380 or a portion thereof.
  • an HSV gB comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 381 or a portion thereof.
  • an antigenic portion of HSV gB comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 382 or a portion thereof.
  • an HSV gB comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 383 or a portion thereof.
  • HSV gB comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 384 or a portion thereof.
  • an HSV gB comprises an amino acid sequence that is identical to the amino acid of SEQ ID NO: 379.
  • an HSV gB comprises an amino acid sequence that is identical to the amino acid of SEQ ID NO: 380.
  • an HSV gB has an amino acid sequence that is identical to the amino acid of SEQ ID NO: 381. In some embodiments, an HSV gB has an amino acid sequence that is identical to the amino acid of SEQ ID NO: 382. In some embodiments, an HSV gB has an amino acid sequence that is identical to the amino acid of SEQ ID NO: 383. In some embodiments, an HSV gB has an amino acid sequence that is identical to the amino acid of SEQ ID NO: 384.
  • HSV compositions e.g., immunogenic compositions, e.g., vaccines
  • HSV gB HSV-1 gB, HSV-2 gB, or both.
  • HSV gB can be unstable.
  • cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane.
  • the viral proteins, including gB undergo substantial structural rearrangements from a metastable prefusion conformation to a stable postfusion conformation (FIG.16).
  • the term “stable”, when applied to glycoprotein B, means that glycoprotein B maintains one of more aspects of a physical structure (e.g., maintains a specific conformation) and/or activity for a specific period of time.
  • a stable glycoprotein B has been modified (e.g., certain mutations) so that its structure is stabilized.
  • a stable glycoprotein B structure is maintained for a specific period of time.
  • a stable glycoprotein B is in a stable prefusion conformation.
  • a stable glycoprotein B is in a stable postfusion conformation. In some embodiments, a stable glycoprotein B maintains a biological relevant activity (e.g., antigenic potential). In some embodiments, a stable glycoprotein B is in a stable prefusion conformation and maintains antigenic potential. [0240] The present disclosure encompasses a recognition that stabilization of gB or antigenic portions thereof can be useful or advantageous for eliciting an immune response.
  • the present disclosure further provides the recognition that stabilization of HSV gB or antigenic portions thereof can be particularly advantageous for use, e.g., in preventing or treating HSV, e.g., in HSV antigen constructs and/or HSV compositions (e.g., immunogenic compositions, e.g., vaccines) as further disclosed herein. Accordingly, the present disclosure provides certain mutations that can stabilize HSV gB or antigenic portions thereof.
  • a polyribonucleotide encoding a GP polypeptide that comprises an HSV glycoprotein B (gB), variant thereof, or one or more antigenic portions thereof.
  • an HSV gB or antigenic portion thereof comprises one or more mutations that stabilize the HSV gB or antigenic portion thereof relative to a comparable HSV gB or antigenic portion thereof that does not comprise the one or more mutations.
  • one or more mutations are one or more amino acid substitutions.
  • one or more amino acid substitutions comprise 120C, 181C, 238C, 251C, 259C, 290C, 291C, 391C, 526C, 571C, 610C, 630C, 636C, 676C, 677C, 680C, 714C, 718C, 725C, 758C, and combinations thereof, wherein the numbering is with reference to SEQ ID NO: 379.
  • an HSV gB comprises an amino acid sequence according to SEQ ID NO: 379 or antigenic portion thereof, except that the HSV gB or antigenic portion thereof comprises one or more mutations comprising 120C, 181C, 238C, 251C, 259C, 290C, 291C, 391C, 526C, 571C, 610C, 630C, 636C, 676C, 677C, 680C, 714C, 718C, 725C, 758C, and combinations thereof.
  • an HSV gB has an amino acid sequence that (i) is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 379 and (ii) comprises one or more mutations, where the one or more mutations comprise 120C, 181C, 238C, 251C, 259C, 290C, 291C, 391C, 526C, 571C, 610C, 630C, 636C, 676C, 677C, 680C, 714C, 718C, 725C, 758C, and combinations thereof.
  • an HSV gB has an amino acid sequence that comprises one or more mutations, where the one or more mutations comprise: (a) 120C and 677C, (b) 181C and 725C, (c) 238C and 610C, (d) 251C and 718C, (e) 259C and 758C, (f) 290C and 680C, (g) 291C and 636C, (h) 391C and 526C, (i) 571C and 676C, (j) 571C and 680C, and/or (k) 630C and 714C, where the numbering is with reference to SEQ ID NO: 379.
  • an HSV gB has an amino acid sequence that (i) is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 379 and (ii) comprises one or more mutations, where the one or more mutations comprise: (a) 120C and 677C, (b) 181C and 725C, (c) 238C and 610C, (d) 251C and 718C, (e) 259C and 758C, (f) 290C and 680C, (g) 291C and 636C, (h) 391C and 526C, (i) 571C and 676C, (j) 571C and 680C, and/or (k) 630C and 714C, where the numbering is with reference to SEQ ID NO: 379.
  • an HSV gB or antigenic portion thereof does not comprise an 516P mutation, where the numbering is with reference to SEQ ID NO: 379.
  • the present disclosure provides a plurality of polyribonucleotides, wherein the plurality of polyribonucleotides comprises a first set of polyribonucleotides that comprises: (i) a polyribonucleotide that encoding an antigenic portion of HSV gC as provided herein, (ii) a polyribonucleotide that encoding an antigenic portion of HSV gD as provided herein, (iii) a polyribonucleotide that encoding an antigenic portion of HSV gE as provided herein, and (iv) a polyribonucleotide that encoding an antigenic portion of HSV gB or variant thereof as provided herein.
  • a first set of polyribonucleotides further comprises a polyribonucleotide encoding an HSV glycoprotein G (gG) or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein H (gH) or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein I (gI) or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein L (gL) or an antigenic portion thereof, or a combination thereof.
  • G HSV glycoprotein G
  • I HSV glycoprotein I
  • gL HSV glycoprotein L
  • combinations provided herein further comprises a second set of polyribonucleotides.
  • a second set of polyribonucleotides encode one or more T-cell string polypeptides.
  • one or more T-cell string polypeptides comprises one or more HSV T-cell antigens or antigenic portions thereof.
  • one or more HSV T-cell antigens comprises (i) one or more HSV RS1 polypeptides or antigenic portions thereof, (ii) one or more HSV RL2 polypeptides or antigenic portions thereof, (iii) one or more HSV UL1 polypeptides or antigenic portions thereof, (iv) one or more HSV UL5 polypeptides or antigenic portions thereof, (v) one or more HSV UL9 polypeptides or antigenic portions thereof, (vi) one or more HSV UL19 polypeptides or antigenic portions thereof, (vii) one or more HSV UL21 polypeptides or antigenic portions thereof, (viii) one or more HSV UL25 polypeptides or antigenic portions thereof, (ix) one or more HSV UL27 polypeptides or antigenic portions thereof, (x) one or more HSV UL29 polypeptides or antigenic portions thereof, (xi) one or more HSV UL30 polypeptides or antigenic
  • Example antigen amino acid sequences are shown in Table 7 below.
  • Table 7 Example amino acid antigen sequences SEQ Antigen Strain Amino Acid Sequence ID NO 476 UL1 HG52 MGFVCLFGLVVMGAWGAWGGSQATEYVLRSVIAKEVGDILRVPCMRTPADDVS F R G G V A G L D G G V A G L D G G V A G L D G A G L D G A P S F A L R Y T T GPULIEGQLGENNELRLTRDALEPCTVGHRRYFIFGGGYVYFEEYAYSHQLSRADVT TVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVI RADANAAMFAGLCAFFEGMGDLGRAVGKVVMGVVGGVVSAVSGVSSFMSNPFGAL G R A P S F A L R Y T T T R A G Y T G V D V A D G E R E I N L E H R A D V A D G E
  • a T-cell string polypeptide includes a plurality of HSV T-cell antigens or antigenic portions thereof (e.g., a plurality of HSV antigens that are or include one or more T cell and/or B cell antigens for HSV).
  • T cell antigens include, e.g., CD4 T cell antigens and/or CD8 T cells.
  • an HSV antigen is a T cell antigen.
  • an HSV antigen is a B cell antigen.
  • an HSV T-cell antigen comprises at least one of UL1, UL21, UL27, UL29, UL39, UL40, UL46, UL47, UL48, UL49, RS1, RL2, UL5, UL9, UL19, UL25, UL30, UL52, US1, US7, US8, UL22, US10, US12, UL26, UL50, and/or UL54 or antigenic portion thereof.
  • a T-cell polypeptide comprises a plurality of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of) UL1, UL21, UL27, UL29, UL39, UL40, UL46, UL47, UL48, UL49, RS1, RL2, UL5, UL9, UL19, UL25, UL30, UL52, US1, US7, US8, UL22, US10, US12, UL26, UL50, and/or UL54 or antigenic portions thereof.
  • a T-cell polypeptide comprises and/or encodes UL54, UL29, UL39, UL9, UL30a (UL30.1), UL40, UL5a (UL5.1), UL21, and/or UL46 or fragments thereof.
  • a T-cell polypeptide comprises and/or encodes UL54, UL29, UL40, and/or UL47 or fragments thereof.
  • a T-cell polypeptide comprises and/or encodes at least UL47 or fragments thereof.
  • a T-cell polypeptide comprises and/or encodes at least UL40 or fragments thereof.
  • a T-cell polypeptide comprises and/or encodes one or more of RL2, UL54, UL9, UL39, UL29, UL5, UL40, UL30, UL49, UL46 and/or UL21 or fragments thereof.
  • the UL1 open reading frame encodes HSV gL (also referred to herein as UL1 polypeptide).
  • an HSV antigen e.g., a T-cell or B cell antigen for HSV
  • a UL1 polypeptide or fragment thereof has at least 80% sequence identity with a UL1 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL1 polypeptides known in the art include UL1 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL1 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 476.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL21 polypeptide or fragment thereof has at least 80% sequence identity with a UL21 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL21 polypeptides known in the art include UL21 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL21 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 477, 478 and/or 479.
  • the UL27 open reading frame encodes HSV gB (also referred to herein as UL27 polypeptide).
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL27 polypeptide or fragment thereof has at least 80% sequence identity with a UL27 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL27 polypeptides known in the art include UL27 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL27 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 480, 481, 482 and/or 483.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL29 polypeptide or fragment thereof has at least 80% sequence identity with a UL29 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL29 polypeptides known in the art include UL29 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL29 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 484, 485, and/or 486.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL39 polypeptide or fragment thereof has at least 80% sequence identity with a UL39 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL39 polypeptides known in the art include UL39 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL39 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 487, 488 and/or 489.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL40 polypeptide or fragment thereof has at least 80% sequence identity with a UL40 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL40 polypeptides known in the art include UL40 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL40 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 490, 491 and/or 492.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL46 polypeptide or fragment thereof has at least 80% sequence identity with a UL46 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL46 polypeptides known in the art include UL46 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL46 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 493, 494 and/or 495.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL47 polypeptide or fragment thereof has at least 80% sequence identity with a UL47 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL47 polypeptides known in the art include UL47 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL47 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 496, 497 and/or 498.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL48 polypeptide or fragment thereof has at least 80% sequence identity with a UL48 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL48 polypeptides known in the art include UL48 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL48 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 499, 500 and/or 501.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL49 polypeptide or fragment thereof has at least 80% sequence identity with a UL49 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL49 polypeptides known in the art include UL49 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL49 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 502, 503 and/or 504.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • RS1 polypeptide or fragment thereof is or includes a RS1 polypeptide or fragment thereof.
  • a RS1 polypeptide or fragment thereof has at least 80% sequence identity with a RS1 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • RS1 polypeptides known in the art include RS1 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a RS1 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 505, 506 and/or 507.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a RL2 polypeptide or fragment thereof has at least 80% sequence identity with a RL2 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • RL2 polypeptides known in the art include RL2 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a RL2 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 508, 509 and/or 510.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL5 polypeptide or fragment thereof has at least 80% sequence identity with a UL5 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL5 polypeptides known in the art include UL5 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL5 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 511, 512 and/or 513.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL9 polypeptide or fragment thereof has at least 80% sequence identity with a UL9 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL9 polypeptides known in the art include UL9 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL9 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 514, 515 and/or 516.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL19 polypeptide or fragment thereof has at least 80% sequence identity with a UL19 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL19 polypeptides known in the art include UL19 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL19 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 517, 518 and/or 519.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL25 polypeptide or fragment thereof has at least 80% sequence identity with a UL25 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL25 polypeptides known in the art include UL25 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL25 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 520, 521 and/or 522.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL30 polypeptide or fragment thereof has at least 80% sequence identity with a UL30 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL30 polypeptides known in the art include UL30 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL30 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 523, 524 and/or 525.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL52 polypeptide or fragment thereof has at least 80% sequence identity with a UL52 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL52 polypeptides known in the art include UL52 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL52 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 526, 527 and/or 528.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a US1 polypeptide or fragment thereof has at least 80% sequence identity with a US1 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • US1 polypeptides known in the art include US1 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a US1 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 532, 533, 534, 535 and/or 536.
  • the US7 open reading frame encodes HSV gI (also referred to herein as US7 polypeptide).
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a US7 polypeptide or fragment thereof has at least 80% sequence identity with a US7 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • US7 polypeptides known in the art include US7 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a US7 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 537, 538, 539 and/or 540.
  • the US8 open reading frame encodes HSV gE (also referred to herein as US8 polypeptide).
  • an HSV antigen is (e.g., a T cell or B cell antigen for HSV) or includes a US8 polypeptide or fragment thereof.
  • a US8 polypeptide or fragment thereof has at least 80% sequence identity with a US8 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • US8 polypeptides known in the art include US8 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a US8 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 541, 542, 543 and/or 544.
  • the UL22 open reading frame encodes HSV gH (also referred to herein as UL22 polypeptide).
  • an HSV antigen is (e.g., a T cell or B cell antigen for HSV) or includes a UL22 polypeptide or fragment thereof.
  • a UL22 polypeptide or fragment thereof has at least 80% sequence identity with a UL22 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL22 polypeptides known in the art include UL22 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL22 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 545, 546, 547 and/or 548.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a US10 polypeptide or fragment thereof has at least 80% sequence identity with a US10 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • US10 polypeptides known in the art include US10 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a US10 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 551, 552, 553 and/or 554.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a US12 polypeptide or fragment thereof has at least 80% sequence identity with a US12 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • US12 polypeptides known in the art include US12 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a US12 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 555.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL26 polypeptide or fragment thereof has at least 80% sequence identity with a UL26 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL26 polypeptides known in the art include UL26 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL26 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 558, 559, 560, and/or 561.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL50 polypeptide or fragment thereof has at least 80% sequence identity with a UL50 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL50 polypeptides known in the art include UL50 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL50 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 556 and/or 557.
  • an HSV antigen e.g., a T cell or B cell antigen for HSV
  • a UL54 polypeptide or fragment thereof has at least 80% sequence identity with a UL54 amino acid sequence set forth in Table 7 or otherwise known in the art, or a corresponding fragment thereof (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity).
  • UL54 polypeptides known in the art include UL54 polypeptides encoded by known HSV strains such as, without limitation, HG52, G, 333, and MS strains.
  • a UL54 polypeptide or fragment thereof has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NOs: 529, 530, and/or 531.
  • at least one of the polypeptides in the second set of polynucleotides encode one or more T-cell string polypeptides.
  • a T-cell string polypeptide can include one or more HSV antigens including one or more T cell antigens (e.g., CD4 and/or CD8 T cell antigens) for HSV of the present disclosure and one or more HSV antigens that is not a T cell antigen of the present disclosure.
  • a polyribonucleotide encoding a T-cell string polypeptide can include and/or encode one or more HSV antigens including one or more B cell antigens for HSV of the present disclosure and one or more HSV antigens that is not a B cell antigen of the present disclosure.
  • a polyribonucleotide encoding a T-cell string polypeptide can include and/or encode one or more HSV antigens including one or more T cell antigens for HSV of the present disclosure and one or more HSV antigens that is a B cell antigen for HSV (e.g., an antigen that is or includes a B cell epitope disclosed herein or otherwise known in the art).
  • a polyribonucleotide encoding a T-cell string polypeptide can include and/or encode one or more HSV antigens including one or more T cell antigens for HSV of the present disclosure and one or more HSV antigens selected from HSV glycoproteins or fragments thereof.
  • a polyribonucleotide encoding a T-cell string polypeptide can include and/or encode one or more HSV antigens including one or more T cell antigens for HSV of the present disclosure and one or more HSV antigens selected from an HSV gD protein or an antigenic fragment thereof, an HSV gB protein or an antigenic fragment thereof, an HSV gE protein or an antigenic fragment thereof, an HSV gG protein or an antigenic fragment thereof, an HSV gI protein or an antigenic fragment thereof, an HSV gH protein or an antigenic fragment thereof, an HSV gL protein or an antigenic fragment thereof, an HSV ICP4 protein or an antigenic fragment thereof, or an ICP8 protein or an antigenic fragment thereof.
  • a polyribonucleotide encoding a T-cell string polypeptide can be present in a composition for delivery of the HSV antigen construct to a subject.
  • an HSV antigen construct can be present in a composition for delivery of one or more HSV antigens and/or epitopes to a subject.
  • a polyribonucleotide encoding a T-cell string polypeptide can be or include an RNA molecule that encodes one or more antigens and/or epitopes.
  • compositions for delivery of a polyribonucleotide encoding a T-cell string polypeptide advantageously include, for example, one or more B cell antigens for HSV and one or more T cell antigens (e.g., CD4 and/or CD8 T cell antigens) for HSV.
  • B cell antigens for HSV and one or more T cell antigens (e.g., CD4 and/or CD8 T cell antigens) for HSV.
  • T cell antigens e.g., CD4 and/or CD8 T cell antigens
  • combination of B cell antigens and T cell antigens can be advantageous in promoting immune system defenses against HSV at multiple lifecycle points include without limitation prior to cellular entry and after cellular entry.
  • the present disclosure provides an insight that many prior strategies for developing pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) for treatment of and/or protection from viral infection have focused primarily, or even almost exclusively, on development of neutralizing antibodies that target surface glycoproteins.
  • the present disclosure identifies a problem with such strategies including, for example, that they may fail to appreciate value or even criticality of ensuring that an induced immune response includes significant T cell activity (in some embodiments, CD4 T cell activity, in some embodiments CD8 T cell activity, in some embodiments, both).
  • the present disclosure provides an insight that consideration of expression of HSV proteins (e.g., at particular periods of the HSV life cycle and/or in particular tissues or compartments of an infected subject) can improve composition (e.g., immunogenic composition, e.g., vaccine) effectiveness.
  • composition e.g., immunogenic composition, e.g., vaccine
  • the present disclosure provides technologies for identifying, selecting, and/or characterizing HSV protein sequences (e.g., HSV-1 protein sequences, HSV-2 protein sequences, or a combination thereof), and combinations thereof, particularly useful for inclusion in pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) as described herein.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • CD4 and CD8 antigen(s) of one or more HSV proteins e.g., HSV-1 proteins, HSV-2 proteins, or a combination thereof
  • HSV proteins e.g., HSV-1 proteins, HSV-2 proteins, or a combination thereof
  • HSV antigen constructs e.g., HSV-1 antigen constructs, HSV-2 antigen constructs, or a combination thereof
  • compositions e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines
  • antigen constructs that induce both neutralizing antibodies and T cells (e.g., CD4 and/or CD8 T cells).
  • neutralizing antibodies and T cells e.g., CD4 and/or CD8 T cells
  • the present disclosure provides such polyribonucleotides and compositions that induce particularly strong neutralizing antibody responses and/or particularly diverse T cell responses (e.g., targeting multiple T cell antigens). [0295] In some embodiments, the present disclosure provides such polyribonucleotides and compositions that induce robust B cell responses. In some embodiments, a B cell response includes the production of a diverse, specific repertoire of antibodies. [0296] In some embodiments, the present disclosure provides such polyribonucleotides and compositions that induce T cell and B cell responses to HSV antigens and/or epitopes.
  • the present disclosure provides the recognition, for example, that polyribonucleotides and compositions comprising RNA molecules as described herein (e.g., encoding for one or more HSV (e.g., HSV-1 and/or HSV-2) antigens and/or epitopes) may result in a higher degree of antigen presentation to various immune system components and/or pathways.
  • administration of such constructs or compositions may induce T cell and/or B cell responses.
  • the present disclosure provides the insight that, e.g., in some embodiments in which T cell and B cell responses are induced in a subject, the subject may have a more sustained, long-term immune response.
  • Such an immune response can be beneficial, e.g., for preventing HSV (e.g., HSV-1 and/or HSV-2) reactivation with a single administration, which may increase vaccination rates and subject compliance as compared with presently available vaccines that require dosing every few years.
  • HSV e.g., HSV-1 and/or HSV-2
  • constructs and compositions comprising RNA molecules as described herein (e.g., encoding for one or more HSV (e.g., HSV-1, HSV- 2, or a combination thereof) antigens and/or epitopes) can provide more diverse protection (e.g., protection against HSV (e.g., HSV-1 and/or HSV-2) variants) because, without wishing to be bound to any particular theory, the constructs and compositions can induce multiple immune system responses.
  • the present disclosure also provides the recognition that, by administering polyribonucleotides and compositions that encode HSV (e.g., HSV-1 and/or HSV-2) antigens and/or epitopes, the polyribonucleotides and compositions described herein avoid administering HSV (e.g., HSV-1 and/or HSV-2) virions, which may infect the subject, go into latency, and reactivate to cause a flare-up.
  • HSV e.g., HSV-1 and/or HSV-2
  • the present disclosure provides an insight (and also identifies a source of a problem in some prior HSV vaccination strategies) that, in some embodiments, particularly effective pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) alter one or more characteristics of the innate immune system.
  • compositions including, for example, compositions that comprise RNA construct(s) encoding HSV (e.g., HSV-1 and/or HSV-2) protein(s) (e.g., HSV antigens or HSV epitopes) as described herein.
  • the present disclosure provides particular pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) formats including, for example, RNA pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) comprising particular elements and/or sequences useful for vaccination.
  • RNA pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • the present disclosure provides a variety of insights and technologies related to such HSV (e.g., HSV-1 and/or HSV-2) antigen constructs and compositions (e.g., RNA vaccine).
  • provided compositions e.g.
  • compositions include an RNA active encoding one or more HSV (e.g., HSV-1 and/or HSV-2) polypeptides or antigenic fragments thereof; in some embodiments such RNA active is a modified RNA format in that its uridine residues are substituted with uridine analog(s) such as pseudouridine; alternatively or additionally, in some embodiments, such RNA active includes particular elements (e.g., cap, 5’UTR, 3’UTR, polyA tail, etc) and/or characteristics (e.g., codon optimization) identified, selected, characterized, and/or demonstrated to achieve significant (e.g., elevated) translatability (e.g., in vitro) and/or expression (i.e., in a subject to whom it has been administered) of encoded protein(s).
  • HSV e.g., HSV-1 and/or HSV-2
  • RNA active is a modified RNA format in that its uridine residues are substituted with uridine analog(s) such as pseudo
  • such RNA active includes particular elements and/or characteristics identified, selected, characterized, and/or demonstrated to achieve significant RNA stability and/or efficient manufacturing, particularly at large scale (e.g., 0.1-10 g, 10-500 g, 500 g-1 kg, 750 g-1.5 kg; those skilled in the art will appreciate that different products may be manufactured at different scales, e.g., depending on patient population size).
  • such RNA manufacturing scale may be within a range of about 0.01 g/hr RNA to about 1 g/hr RNA, 1 g/hr RNA to about 100 g/hr RNA, about 1 g RNA/hr to about 20 g RNA/hr, or about 100 g RNA/hr to about 10,000 g RNA/hr. In some embodiments, such RNA manufacturing scale may be tens or hundreds of milligrams to tens or hundreds of grams (or more) of RNA per batch.
  • such RNA manufacturing scale may allow a batch size within a range of about 0.01 g to about 500 g RNA, about 0.01 g to about 10 g RNA, about 1 g to about 10 g RNA, about 10 g to about 500 g RNA, about 10 g to about 300 g RNA, about 10 g to about 200 g RNA or about 30 g to about 60 g RNA.
  • the present disclosure provides technologies for rapid development of a pharmaceutical composition (e.g., immunogenic composition, e.g., HSV vaccine) for delivering particular HSV (e.g., HSV-1 and/or HSV-2) antigen constructs to a subject.
  • a pharmaceutical composition e.g., immunogenic composition, e.g., HSV vaccine
  • particular HSV e.g., HSV-1 and/or HSV-2 antigen constructs to a subject.
  • the present disclosure provides, for example, nucleic acid constructs encoding HSV (e.g., HSV-1 and/or HSV-2) antigens as described herein, expressed HSV (e.g., HSV-1 and/or HSV-2) proteins, and various methods of production and/or use relating thereto, as well as compositions developed therewith and methods relating thereto.
  • the present disclosure provides technologies for preventing, characterizing, treating, and/or monitoring HSV (e.g., HSV-1 and/or HSV-2) outbreaks and/or infections including, as noted, various nucleic acid constructs and encoded proteins, as well as agents (e.g., antibodies) that bind to such proteins, and compositions that comprise and/or deliver them.
  • HSV e.g., HSV-1 and/or HSV-2
  • agents e.g., antibodies
  • technologies provided herein are designed to augment, induce, promote, enhance and/or improve immunological memory against HSV (e.g., HSV-1 and/or HSV-2) or a component thereof (e.g., a protein or fragment thereof).
  • technologies described herein are designed to act as an immunological boost to a primary composition (e.g., immunogenic composition, e.g., vaccine), such as a composition (e.g., immunogenic composition, e.g., vaccine) directed to an antigen and/or epitopes of HSV (e.g., HSV-1 and/or HSV-2).
  • compositions of the present disclosure comprise one or more polynucleotide constructs (e.g., one or more string constructs) that encode one or more antigens from HSV (e.g., HSV-1 and/or HSV-2).
  • HSV e.g., HSV-1 and/or HSV-2
  • the present disclosure provides compositions comprising nucleic acids encoding such HSV (e.g., HSV-1 and/or HSV-2) antigens; those skilled in the art will appreciate from context when reference to a particular polynucleotide (e.g., a DNA or RNA) as “encoding” such antigens in fact is referencing a coding strand or its complement.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • a subject e.g., a patient
  • related technologies e.g., methods
  • pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • deliver particular HSV-1 antigen constructs to a subject e.g., a patient
  • related technologies e.g., methods
  • the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver particular HSV-2 antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods).
  • pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver particular HSV-1 and HSV-2 antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods).
  • the present disclosure further provides the recognition that some HSV antigens are common to both HSV-1 and HSV-2.
  • HSV antigens include sequences conserved between HSV-1 and HSV-2.
  • HSV-1 antigens have, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to comparable HSV-2 antigens.
  • the present disclosure provides some HSV antigen constructs particularly useful in effective vaccination.
  • HSV antigen constructs are HSV-1 antigen construct, HSV-2 antigen constructs, or a combination thereof.
  • Antigens utilized in accordance with the present disclosure are or include HSV (e.g., HSV-1 and/or HSV-2) components (e.g., antigenic fragments thereof, including epitopes that may comprise non-amino acid, e.g., carbohydrate moieties), which components induce immune responses when administered to humans (or other animals such as rodents and non-human primates susceptible to HSV (e.g., HSV-1 and/or HSV-2) infection).
  • HSV e.g., HSV-1 and/or HSV-2
  • components e.g., antigenic fragments thereof, including epitopes that may comprise non-amino acid, e.g., carbohydrate moieties
  • antigens utilized in provided pharmaceutical compositions include both B-cell and T-cell antigens and/or epitopes, as described herein.
  • delivered antigens include both B-cell and T cell (e.g., CD4 and/or CD8 T cell) antigens and/or epitopes, optionally together in a single antigen polypeptide.
  • antigens utilized in provided pharmaceutical compositions include T cell antigens and/or epitopes.
  • antigens utilized in provided pharmaceutical compositions include B cell, CD4 T cell and CD8 T cell epitopes.
  • the present disclosure defines particularly useful epitopes for inclusion in HSV (e.g., HSV-1 and/or HSV-2) compositions (e.g., immunogenic compositions, e.g., vaccines), and/or provides antigens that include them.
  • HSV HSV-1 and/or HSV-2
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • antigens that include them.
  • Exemplary antigens and/or epitopes for use in compositions described herein included those provided in, e.g., Tables 7 and 8 herein and antigenic fragments thereof.
  • exemplary antigens disclosed in Tables 7 and 8, and/or fragments and/or epitopes thereof can be useful for compositions described herein.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccine
  • comprises or delivers e.g., causes expression of in a recipient organism, for example by administration of a nucleic acid construct, such as an RNA construct as described herein, that encodes it
  • an antigen that is or comprises one or more epitopes (e.g., one or more B-cell and/or one or more T-cell antigens and/or epitopes) of an HSV (e.g., HSV-1 and/or HSV-2) protein.
  • a pharmaceutical composition described herein induces a relevant immune response effective against HSV (e.g., by targeting an HSV-1 protein, an HSV-2 protein, or a combination thereof).
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccine
  • HSV e.g., HSV-1 and/or HSV-2
  • a provided pharmaceutical composition comprises or delivers an antigen that is or comprises a full-length HSV (e.g., HSV-1 and/or HSV-2) protein.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccine
  • HSV e.g., HSV-1 and/or HSV-2 vaccine
  • an antigen that is or comprises a fragment of an HSV (e.g., HSV-1 and/or HSV-2) protein that is less than a full-length HSV (e.g., HSV-1 and/or HSV-2) protein.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccine
  • HSV e.g., HSV-1 and/or HSV-2
  • a chimeric polypeptide that is or comprises part or all of an HSV (e.g., HSV-1 and/or HSV-2) protein and one or more heterologous polypeptide elements.
  • an antigen that is included in and/or delivered by a provided pharmaceutical composition is or comprises one or more peptide fragments of an HSV (e.g., HSV-1 and/or HSV-2) antigen; in some such embodiments, each of the one or more peptide fragments includes at least one epitope (e.g., one or more B cell epitopes and/or one or more T cell epitopes), for example as may be predicted, selected, assessed and/or characterized as described herein.
  • a T-cell string polypeptide that is included in and/or delivered by a provided pharmaceutical composition e.g., immunogenic composition, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccine
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccine
  • HSV e.g., HSV-1 and/or HSV-2
  • HSV e.g., HSV-1 and/or HSV-2
  • a single polypeptide antigen may include a plurality of such fragments, e.g., presented as a string of antigens or fragments thereof as described herein (e.g., in that a single polypeptide includes a plurality of amino acid sequences derived from distinct HSV antigens or fragments thereof, optionally separated by or otherwise associated with amino acid linkers or other intervening or terminal amino acid sequences).
  • a single RNA antigen construct may include a plurality of sequences encoding HSV antigens, e.g., presented as a string of antigen encoding sequences as described herein (e.g., in that a single RNA molecule includes a plurality of nucleic acid sequences encoding distinct HSV antigens or fragments thereof, optionally separated by or otherwise associated with nucleic acid linkers or other intervening or terminal nucleic acid sequences).
  • one or more HSV e.g., HSV-1 and/or HSV-2
  • antigens or antigenic fragments thereof may be linked with one or more sequences with which it is linked in nature.
  • such sequence(s) may be or comprise one or more heterologous elements (e.g., one or more elements, not naturally found in the relevant HSV (e.g., HSV-1 and/or HSV-2) such as a polypeptide or antigenic fragment thereof not naturally found to be directly linked to the relevant HSV (e.g., HSV-1 and/or HSV-2) antigen(s)).
  • heterologous elements e.g., one or more elements, not naturally found in the relevant HSV (e.g., HSV-1 and/or HSV-2) such as a polypeptide or antigenic fragment thereof not naturally found to be directly linked to the relevant HSV (e.g., HSV-1 and/or HSV-2) antigen(s)
  • an antigen peptide provided and/or utilized in accordance with the present disclosure may include one or more linker elements and/or one or more membrane association elements and/or one or more secretion elements, etc.
  • an antigenic polypeptide may comprise a plurality of HSV (e.g., HSV-1 and/or HSV-2) protein fragments or epitopes separated from one another by linkers.
  • HSV e.g., HSV-1 and/or HSV-2
  • an HSV e.g., HSV-1 and/or HSV-2 polypeptide, or fragment or epitope thereof, utilized in a construct as described herein (or encoded by a polyribonucleotide describe herein
  • a utilized antigen may include one or more sequence variations found in circulating strains or predicted to arise, e.g., in light of assessments of sequence conservation and/or evolution of HSV (e.g., HSV-1 and/or HSV-2) polypeptides over time and/or across strains.
  • a utilized antigen may include one or more sequence variations selected, for example, to impact stability, folding, processing and/or display of the antigen or any epitope thereof.
  • an HSV e.g., HSV-1 and/or HSV-2
  • an antigen as described herein shows at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with a relevant corresponding reference (e.g., wild type) polypeptide, fragment or epitope.
  • a relevant corresponding reference e.g., wild type
  • an HSV e.g., HSV-1 and/or HSV-2
  • an antigen as described herein shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology (i.e., identity or conservative substitution as is understood in the art) amino acid sequence identity with a relevant corresponding reference (e.g., wild type) protein, fragment or epitope.
  • sequence homology i.e., identity or conservative substitution as is understood in the art
  • an HSV e.g., HSV-1 and/or HSV-2
  • a relevant corresponding reference e.g., wild type polypeptide, fragment or epitope.
  • assessments of degree of conservation may consider the physiochemical difference between two amino acids as described, for example, in WO2014/180569, which is incorporated herein by reference in its entirety. It is well known in molecular evolution that amino acids that interchange frequently are likely to have chemical and physical similarities whereas amino acids that interchange rarely are likely to have different physico-chemical properties. The likelihood for a given substitution to occur in nature compared with the likelihood for this substitution to occur by chance can measured by log-odds matrices.
  • substitutions with positive T scores would have a lower likelihood of altering immunogenicity.
  • substitutions with negative T scores reflect substitutions that are unlikely to occur in nature and hence correspond to two amino acids that have significantly different physico-chemical properties. Such substitutions would have a greater chance of altering immunogenicity.
  • presence of negative T score substitutions within a sequence may indicate that it would be relatively less useful in a composition antigen as described herein.
  • an HSV antigen construct includes and/or encodes a plurality of HSV antigens (e.g., a plurality of HSV antigens that are or include one or more T cell antigens for HSV) provided in Table 7, or fragments thereof.
  • a plurality of HSV antigens e.g., a plurality of HSV antigens that are or include one or more T cell antigens for HSV
  • an HSV antigen construct can include and/or encode at least one HSV antigen provided in Table 7, or fragments thereof.
  • an HSV antigen construct can include and/or encode at least one T cell antigen for HSV provided in Table 7, or fragments thereof.
  • an HSV antigen construct can include and/or encode a plurality of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) HSV antigens provided in Table 7, or fragments thereof.
  • an HSV antigen construct can include and/or encode a plurality of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) T cell antigens for HSV selected from HSV antigens provided in Table 7, or fragments thereof.
  • an antigen utilized in accordance with the present disclosure includes HSV (e.g., HSV-1 and/or HSV-2) protein sequences identified and/or characterized by one or more of: 1) HLA-I or HLA-II binding (e.g., to HLA allele(s) present in a relevant population) 2) HLA ligandomics data, optionally confirmed by mass spectrometry 3) Relatively high expression 4) Sequence conservation 5) Surface exposure 6) Serum reactivity 7) Immunogenicity (e.g., presence of one or more B-cell and/or T-cell antigens and/or epitopes; evidence of ability to induce sterile protection in model systems including, e.g., humans, non-human primates, and/or mice).
  • HSV e.g., HSV-1 and/or HSV-2
  • HLA-II binding e.g., to HLA allele(s) present in a relevant population
  • HLA ligandomics data optionally confirmed by mass spectrometry
  • HLA-I and/or HLA-II binding is experimentally assessed; in some embodiments it is predicted.
  • predicted HLA-I or HLA-II binding is assessed using an algorithm such as neonmhc 1 and/or neonmhc2, which predict and/or characterize likelihood of MHC class I and MHC class II binding, respectively.
  • an MHC-peptide presentation prediction algorithm or MHC-peptide presentation predictor is or comprises NetMHCpan or NetMHCIIpan.
  • a hidden Markov model approach may be utilized for MHC-peptide presentation prediction and/or characterization.
  • the peptide prediction model MARIA may be utilized.
  • NetMHCpan is not utilized to predict or characterize likelihood of MHC binding for peptides as described herein.
  • the peptide prediction model MARIA may be utilized.
  • NetMHCIIpan is not utilized to predict or characterize likelihood of MHC binding for peptides as described herein.
  • neither NetMHCpan nor NetMHCIIpan is utilized to predict or characterize likelihood of MHC binding for peptides as described herein.
  • an MHC-peptide presentation prediction algorithm or MHC-peptide presentation predictor is or comprises RECON ® (Real-time Epitope Computation for ONcology), which offers high quality MHC-peptide presentation prediction based on expression, processing and binding capabilities. See, for example, Abelin et al., Immunity 21:315, 2017; Abelin et al., Immunity 15:766, 2019. [0329] In some embodiments, HLA binding and/or ligandomics assessments will consider the geographic region of subjects to be immunized. For example, in some embodiments, HLA allelic diversity will be considered.
  • antigen(s) included in a provided pharmaceutical composition will be or comprise peptides (e.g., epitopes) expected or determined, when considered together, to bind to a significant percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of HLA alleles expected or known to be present in a relevant region or population.
  • antigen(s) included in a provided pharmaceutical composition will be or comprise peptides expected or determined, when considered together, to bind to the most prevalent (e.g., the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 most prevalent, or at least 1, 2, 3, 4, or 5 of the 10 most prevalent, etc.) HLA alleles expected or known to be present in a relevant region or population).
  • expression level is experimentally determined (e.g., in a model system or in infected humans). In some embodiments, expression level is a reported level (e.g., in a published or presented report).
  • expression level is assessed as RNA (e.g., via RNASeq). In some embodiments (and typically preferably), expression levels is assessed as protein.
  • sequence conservation is assessed, for example, using publicly available sequence evaluation software (such as, for example, multiple sequence alignment programs MAFFT, Clustal Omega, etc.). In some embodiments, sequence conservation is determined by consultation with published resources (e.g., sequences). In some embodiments, sequence conservation includes consideration of currently or recently detected strains (e.g., in an active outbreak).
  • surface exposure is assessed by reference to publicly available database and/or software.
  • serum reactivity is assessed by contacting serum samples from infected individuals with polypeptides including sequences of interest (e.g., as may be displayed via, for example, phage display or peptide array, etc.; see, for example, Whittemore et al “ A General Method to Discover Epitopes from Sera” PlosOne, 2016; https://doi.org/10.1371/journal.pone.0157462).
  • serum reactivity is assessed by consultation with literature reports and or database data indicating serum-recognized sequences.
  • assessment of immunoreactivity and/or of presence of an epitope may be or comprise consultation with the Immune Epitope Database (IEDB) which those skilled in the art will be aware is a freely available resource funded by NIAID that catalogs experimental data on antibody and T cell epitopes (see iedb.org).
  • IEDB Immune Epitope Database
  • antigen(s) utilized in accordance with the present disclosure are characterized by dendritic cell presentation which, in turn may be indicative of HLA binding and/or of immunogenicity.
  • antigen(s) utilized in accordance with the present disclosure are or comprises sequences (e.g., epitopes, fragments, complete proteins) of HSV proteins found in the HSV envelope.
  • antigen(s) utilized in accordance with the present disclosure are or comprises sequences (e.g., epitopes, fragments, complete proteins) of HSV proteins found in the HSV tegument.
  • sequences e.g., epitopes, fragments, complete proteins
  • the present disclosure provides an insight that, in some embodiments, it may be desirable to include two or more different epitopes, optionally from two or more different HSV (e.g., HSV-1 and/or HSV-2) proteins, in pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) compositions, which can be useful in the treatment of HSV.
  • Table 8 Exemplary antigen fragment Antigen Sequence (Amino Acid) SEQ ID Fragment NO: RL2.1 CTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVN 562 Fragment DPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLS RL2.2 LPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWS 563 Fragment RRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVG RS1 RAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNR 564 Fragment LCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVN AVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGV
  • a polyribonucleotide provided herein encodes one or more of an RL2 polypeptide or antigenic fragment thereof, an RS1 polypeptide or antigenic fragment thereof, a UL54 polypeptide or antigenic fragment thereof, a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, a UL9 polypeptide or antigenic fragment thereof, a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, a UL52 polypeptide or antigenic fragment thereof, a UL1 polypeptide or antigenic fragment thereof, a UL19 polypeptide or antigenic fragment thereof, a UL21 polypeptide or antigenic fragment thereof, a UL27 polypeptide or antigenic fragment thereof, a UL46 polypeptide or antigenic fragment thereof, a UL47
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an RL2 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, a RL2 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL49 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL30 polypeptide or antigenic fragment thereof, a linker, an UL30 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL52 polypeptide or antigenic fragment thereof, a linker, an UL5 polypeptide or antigenic fragment thereof, a linker, an UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL48 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, nucleotide sequences that encode an HSV-1 gD secretory signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or antigenic fragment thereof, and a linker, and a MITD.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, one or more HSV US1 polypeptides or antigenic fragments thereof, one or more HSV US8 polypeptides or antigenic fragments thereof, one or more HSV US12 polypeptides or antigenic fragments thereof, one or more HSV UL50 polypeptides or antigenic fragments thereof, one or more HSV UL26 polypeptides or antigenic fragments thereof, and one or more HSV US10 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an US1 polypeptide or antigenic fragment thereof, a linker, an US1 polypeptide or antigenic fragment thereof, a linker, an US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US12 polypeptide or antigenic fragment thereof, a linker, a UL50 polypeptide or antigenic fragment thereof, a linker, a UL26 polypeptide or antigenic fragment thereof, a linker, a UL26 polypeptide or antigenic fragment thereof, a linker, a US10 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL26 polypeptide or antigenic fragment thereof, a linker, an UL26 polypeptide or antigenic fragment thereof, a linker, an US10 polypeptide or antigenic fragment thereof, a linker, a UL50 polypeptide or antigenic fragment thereof, a linker, a US12 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or antigenic fragment thereof, a linker, a US1 polypeptide or antigenic fragment thereof, a linker, a US1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV UL54 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, and one or more HSV UL21 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an RL2 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, a UL46 polypeptide or antigenic fragment thereof, a linker, a UL21 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL9 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, and one or more HSV UL30 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV UL54 polypeptides or antigenic fragments thereof, one or more HSV UL9 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, and one or more HSV UL5 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an RL2.1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL5.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an RL2.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL5.2 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL30.1 polypeptides or antigenic fragments thereof, and one or more HSV UL29 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV RL2.1 polypeptides or antigenic fragments thereof, one or more HSV UL54 polypeptides or antigenic fragments thereof, one or more HSV UL5.2 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, and one or more HSV UL46 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, a RL2.1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, a RL2.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV RL2.1 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, a RL2.1 polypeptide or antigenic fragment thereof, a linker, a RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL5.1 polypeptides or antigenic fragments thereof, one or more HSV UL5.2 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, and one or more HSV UL30.1 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, and one or more HSV UL21 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL9 polypeptides or antigenic fragments thereof, one or more HSV UL5.1 polypeptides or antigenic fragments thereof, one or more HSV UL5.2 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, and one or more HSV UL30.1 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV RL2.1 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, one or more HSV UL54 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, and one or more HSV UL21 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, a RL2.1 polypeptide or antigenic fragment thereof, a linker, a RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, a RS1 polypeptide or antigenic fragment thereof, a linker, a RL2.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising one or more HSV UL5.1 polypeptides or antigenic fragments thereof, one or more HSV UL5.2 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL30.1 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, and one or more HSV UL21 polypeptides or antigenic fragments thereof.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes a polypeptide comprising, in N-terminus to C-terminus order, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, and a linker.
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is an RL2 polypeptide or antigenic fragment thereof.
  • an RL2 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of CTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPR TAPRSLS (SEQ ID NO: 562).
  • an RL2 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of LPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWN SLWMTPVGNMLFDQGTLVG (SEQ ID NO: 563).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is an RS1 polypeptide or antigenic fragment thereof.
  • an RS1 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of RAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQ GVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVF GPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAAS (SEQ ID NO: 564).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL54 polypeptide or antigenic fragment thereof.
  • a UL54 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of ETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQC YLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCE LTASHTIAPLYVHGKYFYCNSLF (SEQ ID NO: 565).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL29 polypeptide or antigenic fragment thereof.
  • HSV T-cell antigens or antigenic portions thereof e.g., HSV-1 and/or HSV- 2
  • a UL29 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of REDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSRRPPSVQAAAAWAPQGGAGLEAGAR ALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLLGGKNACPLLIFDRTRKFVL (SEQ ID NO: 566).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL39 polypeptide or antigenic fragment thereof.
  • a UL39 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of RTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWM RSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLA (SEQ ID NO: 567).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL49 polypeptide or antigenic fragment thereof.
  • a UL49 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of KMTRGAPKASATPATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFSTAPPSPTAPWTPRVAGFNKRVFCAAVG (SEQ ID NO: 568).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL9 polypeptide or antigenic fragment thereof.
  • a UL9 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of LLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCL FLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDRVLLLHSLTPPGDVTTWG RYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMDGSGARSEPV (SEQ ID NO: 569).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL30 polypeptide or antigenic fragment thereof.
  • HSV T-cell antigens or antigenic portions thereof e.g., HSV-1 and/or HSV- 2
  • a UL30 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of ISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYK VPLDGYGRMNGRGVFRVWDIGQSHF (SEQ ID NO: 570).
  • a UL30 polypeptide or antigenic fragment thereof comprises an amino acid sequence of GLLPCLHVAATVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSM (SEQ ID NO: 571).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL40 polypeptide or antigenic fragment thereof.
  • a UL40 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of TSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHS RVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLR (SEQ ID NO: 572).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL5 polypeptide or antigenic fragment thereof.
  • a UL5 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of HEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEF DEYRRL (SEQ ID NO: 573).
  • a UL5 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of ELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQ HGFMSVVNTNI (SEQ ID NO: 574).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL52 polypeptide or antigenic fragment thereof.
  • a UL52 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of SVAAPVEVTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEGSRALYRDAGGLNGDSFRVTFCLLGT EVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIMALTVAIVHNAPARIGSGSTAPLYEPG ESMRSVV (SEQ ID NO: 575).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL1 polypeptide or antigenic fragment thereof.
  • a UL1 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of RTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETT (SEQ ID NO: 576).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL19 polypeptide or antigenic fragment thereof.
  • a UL19 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of DGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPL HPANLVANTVKRMFHN (SEQ ID NO: 577).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL21 polypeptide or antigenic fragment thereof.
  • a UL21 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of SPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGL (SEQ ID NO: 578).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL27 polypeptide or antigenic fragment thereof.
  • a UL27 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of NYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEV (SEQ ID NO: 579).
  • a UL27 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of SVYPYDEFVLATGDFVYMSPFYGYREGSH (SEQ ID NO: 580).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL46 polypeptide or antigenic fragment thereof.
  • a UL46 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of GLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFKSGA AAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYV (SEQ ID NO: 581).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL47 polypeptide or antigenic fragment thereof.
  • a UL47 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of GPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVL (SEQ ID NO: 582).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL48 polypeptide or antigenic fragment thereof.
  • a UL48 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of ALFNRLLDDLGFSAGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPATRDEL PSYYEAMAQFFRGELRA (SEQ ID NO: 583).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a UL25 polypeptide or antigenic fragment thereof.
  • a UL25 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of FLWEDQTLLRATANTITALAVLRRLLANGNVYADRLDNRLQLGMLIPGAVPAEAIARGASGLDSGAIKSGDNNLEALCVNYVLPLYQA DPTVELTQLFPGLAALCL (SEQ ID NO: 584).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a US1 polypeptide or antigenic fragment thereof.
  • a US1 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of DDASDGWLVDTPPRKSKRPRINLRLTSSPDRRAGVVFPEV (SEQ ID NO: 585).
  • a US1 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of PASLPGIAHAHRRSARQAQMRSGAAWTLDLHYIRQCVNQL (SEQ ID NO: 586).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a US8 polypeptide or antigenic fragment thereof.
  • an US8 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of ILSPTAPSVYPHSEGRKSRRPLTTFGSGSPGRRHSQASYPSVLW (SEQ ID NO: 587).
  • a US8 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of GLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRAPHPAPSARGPL RL (SEQ ID NO: 588).
  • a US8 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of KLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSG LYTLSVVGLSDEARQVASVVLVVEPAP (SEQ ID NO: 589).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is a US12 polypeptide or antigenic fragment thereof.
  • an US12 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of EDREAARTAVTDPELPLLCPPDV (SEQ ID NO: 590).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is an UL50 polypeptide or antigenic fragment thereof.
  • an UL50 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of ANGATVIQPSLRVLRAADGPEACYVLGRSSLNARGLLVMPTRWPSGHACAFVVCNLTGVPVTL (SEQ ID NO: 591).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is an UL26 polypeptide or antigenic fragment thereof.
  • an UL26 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of APLPDRAVPIYVAGFLALYDSGDPGELALDPDTVRAALPPENPLPINVDHRARCEVGRVLAVVNDPRGPFFVGLIACVQLERVLETAAS AAIFERRGPALSREERLLYLITNYLPSVSLSTKRRGDEVPPDRTLFAHVALCAIGRRLGTIVTYDTSLDAA (SEQ ID NO: 592).
  • an UL26 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of HYPPPPAHPYPGMLFAGPSPLEAQIAALVGAIAADRQAGGLPAAAGDHGIRGSAKRRRHEVEQPEYDCG (SEQ ID NO: 593).
  • a polyribonucleotide provided herein encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof (e.g., HSV-1 and/or HSV- 2), wherein at least one antigen is an US10 polypeptide or antigenic fragment thereof.
  • an US10 polypeptide or antigenic fragment thereof comprises or consists of an amino acid sequence of SSPRQRTYVLPRVGIHNAPASDTRAPKRANSRHRADRPPESPGSELYPLNAQALAHLQMLPADHRAFFRTVIEVSRLCALNTHDPPPP LAGARVGQEAQLVHTQWLRANRESSPLWPWRTAAMNFIAAAAPCVQTHRHMHDLLMACAFWC (SEQ ID NO: 594).
  • an HSV e.g., HSV-1 and/or HSV-2
  • T-cell antigen for use in accordance with the present disclosure is an intermediate early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one HSV T-cell antigen comprises an intermediate early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV (e.g., HSV-1 and/or HSV-2) T-cell antigens, wherein each of the HSV T-cell antigens comprises an intermediate early protein or an antigenic fragment thereof.
  • an HSV-2 T-cell antigen for use in accordance with the present disclosure is an intermediate early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an intermediate early protein or an antigenic fragment thereof. In some embodiments, a polyribonucleotide provided herein encodes one or more HSV-2 antigens, wherein each of the HSV-2 antigens comprises an intermediate early protein or an antigenic fragment thereof.
  • an HSV-2 T-cell antigen for use in accordance with the present disclosure comprises an RL2 polypeptide or antigenic fragment thereof, an RS1 polypeptide or antigenic fragment thereof, a UL54 polypeptide or antigenic fragment thereof, or a combination thereof.
  • a polyribonucleotide provided herein encodes one or more of an RL2 polypeptide or antigenic fragment thereof, an RS1 protein or antigenic fragment thereof, and a UL54 protein or antigenic fragment thereof.
  • an HSV e.g., HSV-1 and/or HSV-2
  • T-cell antigen for use in accordance with the present disclosure is an early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV (e.g., HSV-1 and/or HSV-2) T-cell antigens, wherein at least one HSV T-cell antigen comprises an early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV (e.g., HSV-1 and/or HSV-2) T-cell antigens, wherein each of the HSV T-cell antigens comprises an early protein or an antigenic fragment thereof.
  • HSV e.g., HSV-1 and/or HSV-2
  • an HSV (e.g., HSV-1, HSV-2, or a combination) T-cell antigen for use in accordance with the present disclosure is an early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV T-cell antigens, wherein at least one HSV T-cell antigen comprises an early protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV T-cell antigens, wherein each of the HSV T-cell antigens comprises an early protein or an antigenic fragment thereof.
  • the HSV T-cell antigen is a HSV-2 T-cell antigen.
  • an HSV (e.g., HSV-1, HSV-2, or a combination) T-cell antigen for use in accordance with the present disclosure comprises a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, a UL9 polypeptide or antigenic fragment thereof, or a combination thereof.
  • a polyribonucleotide encoding a T-cell string encodes a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, and a UL9 polypeptide or antigenic fragment thereof.
  • an HSV e.g., HSV-1, HSV-2, or a combination
  • a polyribonucleotide encoding a T-cell string encodes one or more of a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, and a UL52 polypeptide or antigenic fragment thereof.
  • an HSV e.g., HSV-1, HSV-2, or a combination
  • a polyribonucleotide encoding a T-cell string encodes one or more of a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, a UL9 polypeptide or antigenic fragment thereof, a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, and a UL52 polypeptide or antigenic fragment thereof.
  • an HSV (e.g., HSV-1 and/or HSV-2) antigen for use in accordance with the present disclosure is a late protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one HSV antigen comprises a late protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein each of the HSV antigens comprises a late protein or an antigenic fragment thereof.
  • an HSV (e.g., HSV-1, HSV-2, or a combination) T-cell antigen for use in accordance with the present disclosure is a late protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV antigens, wherein at least one HSV antigen comprises a late protein or an antigenic fragment thereof.
  • a polyribonucleotide provided herein encodes one or more HSV antigens, wherein each of the HSV antigens comprises a late protein or an antigenic fragment thereof.
  • an HSV T-cell antigen is an HSV-2 T-cell antigen.
  • an HSV e.g., HSV-1, HSV-2, or a combination
  • a polyribonucleotide encoding a T-cell string encodes one or more of a UL1 polypeptide or antigenic fragment thereof, a UL19 polypeptide or antigenic fragment thereof, a UL21 polypeptide or antigenic fragment thereof, a UL27 polypeptide or antigenic fragment thereof, a UL46 polypeptide or antigenic fragment thereof, a UL47 polypeptide or antigenic fragment thereof, a UL48 polypeptide or antigenic fragment thereof, and a UL25 polypeptide or antigenic fragment thereof.
  • Exemplary polyribonucleotide sequences encoding antigenic fragments are examples of a UL1 polypeptide or antigenic fragment thereof, a UL19 polypeptide or antigenic fragment thereof, a UL21 polypeptide or antigenic fragment thereof, a UL27 polypeptide or antigenic fragment thereof, a UL46 polypeptide or antigenic fragment thereof, a UL47 polypeptide or antigenic fragment thereof, a UL48 polypeptide
  • Table 9 Exemplary polyribonucleotide sequences encoding antigenic fragments SEQ ID Antigen Optimized Nucleotide Sequence NO c c g a a a g c gc c c g a a a g c gc uccaggcgauacuggaacuccugcuccugccucuggcgagaucgccccuccuaauucuacaagaagc gccagcgagagccggcacagaugauaa 611 US7 [HSV-2 Opt3 gguccuaccgugucucucgugucugacagccugguagacgccggugcugugggcccucagggcuuc a a cc cg a ac gg g u a c c u a u c c u a u g c a u g c a u g c a
  • a first set of polyribonucleotides further comprises a polyribonucleotide encoding an HSV glycoprotein B (gB) , variant thereof, or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein G (gG) or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein H (gH) or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein I (gI) or an antigenic portion thereof, a polyribonucleotide encoding an HSV glycoprotein L (gL) or an antigenic portion thereof, or a combination thereof.
  • gB HSV glycoprotein B
  • additional polyribonucleotides are used with the first and/or second set of polyribonucleotides.
  • an additional polyribonucleotide encodes a polypeptide comprising ICP0, ICP4, ICP22, VP16, ICP47, VHS, or US3.
  • an additional set of polyribonucleotides encodes a set of polypeptides, wherein the set of polypeptides comprises ICP0, ICP4, ICP22, VP16, ICP47, VHS, US3, or any combination thereof.
  • an additional set of polyribonucleotides encodes a set of polypeptides, wherein the set of polypeptides comprises ICP0, ICP4, or both.
  • a first set of polyribonucleotides comprises: a polyribonucleotide encoding an HSV glycoprotein B (gB), variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV glycoprotein C (gC) , variant thereof, or an antigenic portion thereof; and a polyribonucleotide encoding an HSV glycoprotein D (gD) , variant thereof, or an antigenic portion thereof.
  • an additional set of polyribonucleotides encodes a set of polypeptides, wherein the set of polypeptides comprises ICP0, ICP4, or both.
  • a combination provided herein comprises a polyribonucleotide encoding an HSV glycoprotein B (gB), variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV glycoprotein C (gC) , variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV glycoprotein D (gD) , variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV ICP0, variant thereof, or an antigenic portion thereof; and a polyribonucleotide encoding an HSV ICP4, variant thereof, or an antigenic portion thereof.
  • a combination provided herein comprises a polyribonucleotide encoding an antigenic portion HSV glycoprotein B (gB), variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV glycoprotein C (gC) , variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV glycoprotein D (gD) , variant thereof, or an antigenic portion thereof; a polyribonucleotide encoding an HSV ICP0, variant thereof, or an antigenic portion thereof; and a polyribonucleotide encoding an HSV ICP4, variant thereof, or an antigenic portion thereof.
  • gB HSV glycoprotein B
  • gC HSV glycoprotein C
  • gD HSV glycoprotein D
  • a combination provided herein comprises a polyribonucleotide encoding an antigenic portion HSV glycoprotein B (gB); a polyribonucleotide encoding an antigenic portion of HSV glycoprotein C (gC); a polyribonucleotide encoding an HSV glycoprotein D (gD) or an antigenic portion thereof; a polyribonucleotide encoding an antigenic portion of HSV ICP0; and a polyribonucleotide encoding an antigenic portion of HSV ICP4.
  • a combination provided herein comprises a polyribonucleotide encoding an antigenic portion HSV-2 glycoprotein B (gB); a polyribonucleotide encoding an antigenic portion of HSV-2 glycoprotein C (gC); a polyribonucleotide encoding an HSV-2 glycoprotein D (gD) or an antigenic portion thereof; a polyribonucleotide encoding an antigenic portion of HSV-2 ICP0; and a polyribonucleotide encoding an antigenic portion of HSV-2 ICP4. D.
  • polyribonucleotides encoding polypeptides (e.g., GP polypeptide) comprising (i) an HSV glycoprotein or antigenic portion thereof and (ii) a secretory signal.
  • polyribonucleotides encoding polypeptides (e.g., T-cell string polypeptides) comprising (i) one or more HSV T-cell antigens or antigenic portions thereof and (ii) secretory signal.
  • a GP polypeptide comprises a secretory signal.
  • a GP polypeptide comprises (i) an HSV gC or antigenic portion thereof and (ii) a secretory signal. In some embodiments, a GP polypeptide comprises (i) an HSV gD or antigenic portion thereof and (ii) a secretory signal. In some embodiments, a GP polypeptides comprises (i) an HSV gD or antigenic portion thereof and (ii) a secretory signal. In some embodiments, a GP polypeptide comprises (i) an HSV gB, variant thereof, or one or more antigenic portions thereof and (ii) a secretory signal. [0435] In some embodiments, a T-cell string polypeptide comprises a secretory signal.
  • a T-cell string polypeptide comprises (i) one or more HSV T-cell antigens or antigenic portions thereof and (ii) a secretory signal.
  • a secretory signal is functional in mammalian cells.
  • a utilized secretory signal is a heterologous secretory signal.
  • a secretory signal comprises or consists of a human secretory signal.
  • a secretory signal comprises or consists of an IL2 secretory signal.
  • a secretory signal comprises or consists of a viral secretory signal.
  • a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal).
  • a secretory signal comprises or consists of an HSV-1 secretory signal.
  • a secretory signal comprises or consists of an HSV-2 secretory signal.
  • an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal (e.g., an HSV-1 or HSV-2 gD secretory signal).
  • gD HSV glycoprotein D
  • an HSV secretory signal comprises or consists of an HSV-1 gD secretory signal.
  • an HSV-1 gD secretory signal comprises one or more additional amino acids. In some embodiments, an HSV-1 gD secretory signal comprises KY at the C terminus of the signal sequence. In some embodiments, an HSV secretory signal comprises or consists of an HSV-2 gD secretory signal. In some embodiments, an HSV-2 gD secretory signal comprises one or more additional amino acids. In some embodiments, an HSV-2 gD secretory signal comprises KY, KYA, KYAL (SEQ ID NO: 842), or KYALA (SEQ ID NO: 841) at the C terminus of the signal sequence.
  • an HSV secretory signal comprises or consists of an HSV glycoprotein C (gC) secretory signal (e.g., an HSV-1 or HSV-2 gC secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV-2 gC secretory signal. [0440] In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein E (gE) secretory signal (e.g., an HSV-1 or HSV-2 gE secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV-1 gE secretory signal.
  • gE HSV glycoprotein E secretory signal
  • an HSV secretory signal comprises or consists of an HSV-2 gE secretory signal. In some embodiments, an HSV-2 gE secretory signal comprises one or more additional amino acids. In some embodiments, an HSV-2 gE secretory signal comprises RTS. In some embodiments, an HSV-2 secretory signal comprises A20V, A21V, A22V substitutions. [0441] In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein B (gB) secretory signal (e.g., an HSV-1 or HSV-2 gB secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV-1 gB secretory signal.
  • gB HSV glycoprotein B
  • an HSV secretory signal comprises or consists of an HSV-1 gB secretory signal.
  • an HSV-1 gB secretory signal comprises one or more additional amino acids.
  • an HSV-1 gB secretory signal comprises AP at the C terminus of the signal sequence.
  • an HSV secretory signal comprises or consists of an HSV-2 gB secretory signal.
  • an HSV secretory signal comprises or consists of an HSV glycoprotein I (gI) secretory signal (e.g., an HSV-1 or HSV-2 gI secretory signal).
  • an HSV secretory signal comprises or consists of an HSV-1 gI secretory signal.
  • an HSV-1 gI secretory signal comprises one or more additional amino acids.
  • an HSV secretory signal comprises or consists of an HSV- 2 gI secretory signal.
  • an HSV-2 gI secretory signal comprises an additional leucine residue at the C terminus of the signal sequence.
  • a secretory signal comprises or consists of an Ebola spike glycoprotein (EboZ).
  • EboZ secretory signal comprises one or more additional amino acids.
  • an EboZ secretory signal comprises IP at the C terminus of the signal sequence.
  • a secretory signal is characterized by a length of about 15 to 30 amino acids.
  • a secretory signal is positioned at the N-terminus of a polyribonucleotide.
  • a secretory signal preferably allows transport of a polyribonucleotide with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosomal- lysosomal compartment.
  • polyribonucleotides comprising an HSV antigen do not comprise a secretory signal.
  • polyribonucleotides comprising an HSV antigen further comprise a codon initiation start site.
  • a secretory signal is one listed in Table 10, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto.
  • a secretory signal is selected from those included in the Table 10 below and/or those encoded by the sequences in Table 11 and/or Table 12 below.
  • a polypeptide (e.g., a GP polypeptide or a T-cell string polypeptide) described herein includes a transmembrane region.
  • a polyribonucleotide described herein encodes a polypeptide (e.g., a GP polypeptide or a T-cell string polypeptide) that comprises a transmembrane region.
  • a transmembrane region is located at the N-terminus of a polypeptide (e.g., a GP polypeptide or a T-cell string polypeptide).
  • a transmembrane region is located at the C-terminus of a polypeptide (e.g., a GP polypeptide or a T-cell string polypeptide). In some embodiments, a transmembrane region is not located at the N-terminus or C-terminus of a polypeptide (e.g., a GP polypeptide or a T-cell string polypeptide). In some embodiments, a polypeptide does not include a transmembrane region. [0449] Transmembrane regions are known in the art, any of which can be utilized in a polypeptide described herein.
  • a transmembrane region comprises or is a transmembrane region of Hemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV-G), Rabies virus, or a seven transmembrane domain receptor.
  • HA Hemagglutinin
  • EIAV equine infectious anaemia virus
  • MMV murine leukaemia virus
  • VSV-G G protein of vesicular stomatitis virus
  • Rabies virus or a seven transmembrane domain receptor.
  • a polypeptide comprises an HSV transmembrane region.
  • an HSV transmembrane region is an HSV-1 or HSV-2 transmembrane region.
  • an HSV transmembrane region is an HSV-2 gD transmembrane region. In some embodiments, an HSV transmembrane region is an HSV-2 gC transmembrane region. In some embodiments, an HSV transmembrane region is an HSV-2 gE transmembrane region. In some embodiments, an HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 468).
  • a utilized transmembrane region is a heterologous transmembrane region.
  • a heterologous transmembrane region comprises or consists of a human transmembrane region.
  • a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region.
  • hDAF-GPI anchor region comprises or consists of an amino acid sequence of PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 469).
  • an HSV transmembrane region comprises or consists of an HSV gB transmembrane region, e.g., comprising or consisting of an amino acid sequence of MSNPFGALAVGLLVLAGLAAAFFAFRYVMRL (SEQ ID NO: 470) or MSNPFGALAVGLLVLAGLVAAFFAFRYVLQL (SEQ ID NO: 471).
  • a viral transmembrane region comprises or consists of a vesicular stomatitis virus G (VSV-G) transmembrane region.
  • a VSV-G transmembrane region comprises or consists of an amino acid sequence of IASFFFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMN (SEQ ID NO: 245).
  • Example transmembrane regions are provided in the following Table 13: Table 13: Example transmembrane regions SEQ Transmembrane Region Sequence (Amino Acid) ID 468 HSV-1 gD GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR 469 hDAF-GPI anchor region PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT [0453] Provided herein are polypeptides (e.g., GP polypeptides and/or T-cell string polypeptides) that comprise a trafficking signal.
  • polypeptides e.g., GP polypeptides and/or T-cell string polypeptides
  • a trafficking signal is an MHC Class I Trafficking Signal (MITD).
  • MITD MHC Class I Trafficking Signal
  • at least one of the one or more GP polypeptides comprise a MITD.
  • at least one of the one or more GP polypeptides comprise (i) an HSV gC or antigenic portion thereof and (ii) a MITD.
  • at least one of the one or more GP polypeptides comprise (i) an HSV gD or antigenic portion thereof and (ii) a MITD.
  • at least one of the one or more GP polypeptides comprise (i) an HSV gD or antigenic portion thereof and (ii) a MITD.
  • At least one of the one or more GP polypeptides comprise (i) an HSV gB, variant thereof, or one or more antigenic portions thereof and (ii) a MITD.
  • at least one of the one or more T-cell string polypeptides comprises a MITD.
  • a MITD comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 473).
  • a MITD comprises or consists of an amino acid sequence of IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 473).
  • G. Multimerization Regions Provided herein are polypeptides (e.g., GP polypeptides and/or T-cell string polypeptides) that comprise one or more multimerization regions.
  • a multimerization region is a heterologous multimerization region.
  • a heterologous multimerization region comprises a dimerization, trimerization or tetramerization region.
  • At least one of the one or more GP polypeptides comprise one or more multimerization regions. In some embodiments, at least one of the one or more GP polypeptides comprise (i) an HSV gC or antigenic portion thereof and (ii) one or more multimerization regions. In some embodiments, at least one of the one or more GP polypeptides comprise (i) an HSV gD or antigenic portion thereof and (ii) one or more multimerization regions. In some embodiments, at least one of the one or more GP polypeptides comprise (i) an HSV gD or antigenic portion thereof and (ii) one or more multimerization regions.
  • At least one of the one or more GP polypeptides comprise (i) an HSV gB, variant thereof, or one or more antigenic portions thereof and (ii) one or more multimerization regions.
  • at least one of the one or more T-cell string polypeptides comprises one or more multimerization regions.
  • a multimerization region is one described in WO2017/081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1116-1167, or fragments or variants thereof).
  • Example trimerization and tetramerization regions include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53.
  • a multimerization region comprising or consisting of the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 474).
  • a GP polypeptide comprising an HSV-2 gB or antigenic portion thereof, as described herein, is able to form a trimeric complex.
  • a GP polypeptide comprising an HSV-2 gB or antigenic portion thereof, as described herein may further comprise a multimerization region allowing formation of a multimeric complex, such as for example a trimeric complex of an HSV-2 gB described herein.
  • a multimerization region allowing formation of a multimeric complex comprises a trimerization region, for example, a trimerization region described herein.
  • a GP polypeptide comprising an HSV-2 gB or antigenic portion thereof, as described herein further includes a T4-fibritin-derived “foldon” trimerization region, for example, to increase its immunogenicity.
  • a polypeptide (e.g., a GP polypeptide and/or T-cell string polypeptide) includes one or more linkers. In some embodiments, at least one of the one or more T-cell string polypeptides comprises a secretory signal. [0464] In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids.
  • G glycine
  • a linker comprises one or more serine (S) amino acids.
  • a linker comprises a glycine-serine linker.
  • a “glycine-serine linker” as used herein refers to a linker that comprises predominantly (e.g., 80% or more) glycine and serine amino acids.
  • a linker includes amino acids selected based on a cleavage predictor to generate highly-cleavable linkers.
  • a linker is or comprises SGGGGSGGGGS (SEQ ID NO: 475).
  • a linker is or comprises GSPGSGSGS (SEQ ID NO: 476).
  • a linker is or comprises GGSGGGGSGG (SEQ ID NO: 477). In some embodiments, a linker is one presented in Table 14. In some embodiments, a linker is or comprises a sequence as set forth in WO2017/081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof). [0466] In some embodiments, a GP polypeptide described herein comprises a linker between a C-terminal region or fragment thereof and a transmembrane region. In some embodiments, a GP polypeptide described herein comprises a linker after a minor repeat sequence.
  • a T-cell string polypeptide comprises one or more linkers. In some embodiments, a T-cell string polypeptide comprises a one or more linkers separating one or more HSV T-cell antigens or antigenic portions thereof.
  • Example linkers are provided in the following Table 14: Table 14: Example linkers SEQ ID NO: Sequence (Amino Acid) 475 SGGGGSGGGGS [0468]
  • a polyribonucleotide encodes a GP polypeptide, wherein the GP polypeptide comprises an HSV glycoprotein or antigenic portion thereof and a secretory signal.
  • a polyribonucleotide encodes a GP polypeptide, wherein the GP polypeptide comprises an antigenic portion of an HSV glycoprotein and a secretory signal.
  • Example polyribonucleotide constructs encoding a gC, gD, or gE as described herein are provided in Table 15 below.
  • Example combinations of an antigenic portion of HSV-2 gC and a secretory signal are provided in Table 16 below, along with example corresponding amino acid sequences.
  • a GP polypeptide as described herein (or encoded by a polyribonucleotide as described herein) comprises an antigenic portion of HSV-2 gD and a secretory signal.
  • Example combinations of an antigenic portion of HSV-2 gD and a secretory signal are provided in Table 16 below, along with example corresponding amino acid sequences.
  • a GP polypeptide as described herein (or encoded by a polyribonucleotide as described herein) comprises an antigenic portion of HSV-2 gE and a secretory signal.
  • Example combinations of an antigenic portion of HSV-2 gE and a secretory signal are provided in Table 16 below, along with example corresponding amino acid sequences.
  • Example GP polypeptides comprising a secretory signal and an antigenic portions of an HSV-2 glycoprotein SEQ ID Secretory Antigenic NO: signal fragment Sequence (Amino acids) T IR P R W R SS PR RY Q Y G T A IP S DP PP Q T PP G YP P S LE A G PE G A PL P Q T YYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPP RTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEG VTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYP S LA G G YY T TF GI S C V E T G G C P V D A L L E P A G G YY T TF GI SN N N L TC G C RL 69 IL2 HSV-2 gD MRMQLLLLIALSLAL
  • a pol ribonucleotide encoding a T-cell string can include, in ).
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 595.
  • a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 693 of amino acid sequence SEQ ID NO: 595.
  • a polyribonucleotide encoding a T-cell string can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL29 polypeptide or antigenic ).
  • a polyribonucleotide encodes a polypeptide having an amino acid 26- can YG RAY FL FAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECD SIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRGGSGGGGSGGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTR LFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGS PRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGSVAAPVE VTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSD
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 597. [0479] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 792 of amino acid sequence SEQ ID NO: 597.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL1 polypeptide or antigenic fragment thereof, a linker, an UL19 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL46 polypeptide or fragment thereof, a linker, a UL47 polypeptide or fragment thereof, a linker, a UL25 polypeptide or fragment thereof, a linker, a UL48 polypeptide or fragment thereof, a linker, and a MITD (see FIG.18D).
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGRTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDL SHSVFPADTQETTGGSGGGGSGGDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQ NMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVKRMFHNGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQR HGLYVPAPDEPTLADAMNGLGGSGGGGSGGNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEE VGGSGGGGSGGSVYPYDEFVLATGDFVYMSPFYGYREGSHG
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 598.
  • a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 858 of amino acid sequence SEQ ID NO: 598.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, a RL2 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLR PQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYI PGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDL AAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLI VVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGV
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 599. [0483] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 692 of amino acid sequence SEQ ID NO: 599.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL49 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGLLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCS LRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVAR FCRQFTDRVLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMD GSGARSEPVGGSGGGGSGGKMTRGAPKASATPATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFSTAPPSPTAPW TPRVAGFNKRVFCAAVGGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLD
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 600. [0485] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 722 of amino acid sequence SEQ ID NO: 600.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL52 polypeptide or antigenic fragment thereof, a linker, an UL5 polypeptide or antigenic fragment thereof, a linker, an UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or fragment thereof, a linker, a UL30 polypeptide or fragment thereof, a linker, a UL30 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGSVAAPVEVTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEG SRALYRDAGGLNGDSFRVTFCLLGTEVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIM ALTVAIVHNAPARIGSGSTAPLYEPGESMRSVVGGSGGGGSGGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWT RLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTG SPRGGLMSVALQTDNYTLMGYTYTRVFAEELRRRHATAGVAEFLEESPLPY
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 601. [0487] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 792 of amino acid sequence SEQ ID NO: 601.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL48 polypeptide or antigenic fragment thereof, a linker, an UL25 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, a UL46 polypeptide or fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL21 polypeptide or fragment thereof, a linker, a UL19 polypeptide or fragment thereof, a linker, a UL1 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGALFNRLLDDLGFSAGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHV PERSPIDIRAHGDVAFPTLPATRDELPSYYEAMAQFFRGELRAGGSGGGGSGGFLWEDQTLLRATANTITALAVLRRLLANGNVYAD RLDNRLQLGMLIPGAVPAEAIARGASGLDSGAIKSGDNNLEALCVNYVLPLYQADPTVELTQLFPGLAALCLGGSGGGGSGGGPDAA VFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGGLASDPHYDYIR HYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLL
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 602. [0489] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 858 of amino acid sequence SEQ ID NO: 602.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-2 gD secretory signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGRLTSGVGTAALLVVAVGLRVVCACTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQ TGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPD PEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFA GAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRR
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 603. [0491] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 693 of amino acid sequence SEQ ID NO: 603.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-2 gD secretory signal, an UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, a RL2 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGRLTSGVGTAALLVVAVGLRVVCAETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLR PQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYI PGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDL AAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLI VVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGV
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 604. [0493] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 692 of amino acid sequence SEQ ID NO: 604.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an US1 polypeptide or antigenic fragment thereof, a linker, an US1 polypeptide or antigenic fragment thereof, a linker, an US8 polypeptide or antigenic fragment thereof, a linker, a US8 polypeptide or fragment thereof, a linker, a US8 polypeptide or fragment thereof, a linker, a US12 polypeptide or fragment thereof, a linker, a UL50 polypeptide or fragment thereof, a linker, a UL26 polypeptide or fragment thereof, a linker, a UL26 polypeptide or fragment thereof, a linker, a US10 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGDDASDGWLVDTPPRKSKRPRINLRLTSSPDRRAGVVFPEVGGSGGGGSGGPASLPGIAHAH RRSARQAQMRSGAAWTLDLHYIRQCVNQLGGSGGGGSGGILSPTAPSVYPHSEGRKSRRPLTTFGSGSPGRRHSQASYPSVLWGG SGGGGSGGGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRAPHP APSARGPLRLGGSGGGGSGGKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWR DRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPGGSGG
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 605. [0495] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 905 of amino acid sequence SEQ ID NO: 605.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL26 polypeptide or antigenic fragment thereof, a linker, an UL26 polypeptide or antigenic fragment thereof, a linker, an US10 polypeptide or antigenic fragment thereof, a linker, a UL50 polypeptide or fragment thereof, a linker, a US12 polypeptide or fragment thereof, a linker, a US8 polypeptide or fragment thereof, a linker, a US8 polypeptide or fragment thereof, a linker, a US8 polypeptide or fragment thereof, a linker, a US8 polypeptide or fragment thereof, a linker, a US1 polypeptide or fragment thereof, a linker, a US1 polypeptide or fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGHYPPPPAHPYPGMLFAGPSPLEAQIAALVGAIAADRQAGGLPAAAGDHGIRGSAKRRRHEVE QPEYDCGGGSGGGGSGGAPLPDRAVPIYVAGFLALYDSGDPGELALDPDTVRAALPPENPLPINVDHRARCEVGRVLAVVNDPRGPF FVGLIACVQLERVLETAASAAIFERRGPALSREERLLYLITNYLPSVSLSTKRRGDEVPPDRTLFAHVALCAIGRRLGTIVTYDTSLDAA GGSGGGGSGGSSPRQRTYVLPRVGIHNAPASDTRAPKRANSRHRADRPPESPGSELYPLNAQALAHLQMLPADHRAFFRTVIEVSRL CALNTHDPPPPLAGARVGQEAQLVHTQWLRANRESSPLWPWRTAAMNFIAAAAPCVQ
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 606. [0497] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 905 of amino acid sequence SEQ ID NO: 606.
  • a polyribonucleotide encoding a T-cell string can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or fragment thereof, a linker, and an HSV-1 transmembrane region.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQ TGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPD PEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFA GAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDL
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 607. [0499] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 693 of amino acid sequence SEQ ID NO: 607.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or fragment thereof, a linker, and a VSV-G transmembrane region.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQ TGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPD PEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFA GAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDL
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 608. [0501] In some embodiments, a polyribonucleotide encodes a T-cell string that comprises amino acids 26- 693 of amino acid sequence SEQ ID NO: 608.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASA DETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVS EIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGGPDAAVFRSSL GSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGR
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 633. [0503] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-651 of amino acid sequence SEQ ID NO: 633.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGG SGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFK SGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGGPDA AVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGETLVAHGPSLY RTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQD
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 634. [0505] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-651 of amino acid sequence SEQ ID NO: 634.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGREDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSR RPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASL LGGKNACPLLIFDRTRKFVLGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPL VRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYL KRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGLLNNYDVLVLDEVMSTLGQLYSPTMQQQ
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 635. [0507] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-1156 of amino acid sequence SEQ ID NO: 635.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLG GLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFA ASFAAIAYLRTNNLLRGGSGGGGSGGISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQY GPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCE LLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAEE
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 636. [0509] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-1156 of amino acid sequence SEQ ID NO: 636.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an RL2.1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL5.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASA DETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVS EIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGLLNNYDVLVLD EVMSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLC
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 637. [0511] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-880 of amino acid sequence SEQ ID NO: 637.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an RL2.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHA YLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLRE YATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVER GLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGLLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRLLR TCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMP
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 638. [0513] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-880 of amino acid sequence SEQ ID NO: 638.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPRE AAFAGRVLGGSGGGGSGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHV LLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKY VGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGGSGGELFGEVFESA PFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAEELRRRH
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 639. [0515] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-927 of amino acid sequence SEQ ID NO: 639.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGREDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSR RPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASL LGGKNACPLLIFDRTRKFVLGGSGGGGSGGISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTF VKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGTSQCPDINHLRSLSILNRWL ETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKD
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 640. [0517] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-927 of amino acid sequence SEQ ID NO: 640.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, a RL2.1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASA DETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVS EIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGELFGEVFESAP FSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAEELRRR
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, a RL2.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDC DPSLHVLLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLW TADKYVGGSGGGGSGGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVL GGSGGGGSGGTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYY VEQKDILHYY VEQKDILHYY VEQKDILHYY VEQKDILHYY VEQKDILHYY
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 642. [0521] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-874 of amino acid sequence SEQ ID NO: 642.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, a RL2.1 polypeptide or antigenic fragment thereof, a linker, a RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWS AERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADG PVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVP MSPREYRRAVLPALDGRAAASGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQEN
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 643. [0523] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-587 of amino acid sequence SEQ ID NO: 643.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGREDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSR RPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASL LGGKNACPLLIFDRTRKFVLGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPL VRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYL KRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGLLNNYDVLVLDEVMSTLGQLYSPTMQQQ
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 644. [0525] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-638 of amino acid sequence SEQ ID NO: 644.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHA YLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYT LMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGTSQCPDINHLRSLSILNRWLETEL VFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVAR TINHPAIRVKVDWLEARVRECDSIPEKFILMILIEG
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 645. [0527] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-543 of amino acid sequence SEQ ID NO: 645.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPRE AAFAGRVLGGSGGGGSGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHV LLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKY VGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGGSGGIVGIVAGLAVL AVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 646. [0529] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-335 of amino acid sequence SEQ ID NO: 646.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL29 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGREDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSR RPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASL LGGKNACPLLIFDRTRKFVLGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPL VRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYL KRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGLLNNYDVLVLDEVMSTLGQLYSPTMQQQ
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 647. [0531] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-1156 of amino acid sequence SEQ ID NO: 647.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYG PEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGTSQCPDINHLRSLSILNRWLETELVF VGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTI NHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCE LLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFA
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 648. [0533] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-1156 of amino acid sequence SEQ ID NO: 648.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, a RL2.1 polypeptide or antigenic fragment thereof, a linker, a RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWS AERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADG PVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVP MSPREYRRAVLPALDGRAAASGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQEN
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 649. [0535] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-897 of amino acid sequence SEQ ID NO: 649.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, a RS1 polypeptide or antigenic fragment thereof, a linker, a RL2.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGG SGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFK SGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGGPDA AVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGETLVAHGPSLY RTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQD
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 650. [0537] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-897 of amino acid sequence SEQ ID NO: 650.
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL5.1 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL21 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHA YLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYT LMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGTSQCPDINHLRSLSILNRWLETEL VFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVAR TINHPAIRVKVDWLEARVRECDSIPEKFILMILIEG
  • a polyribonucleotide encoding a T-cell string polypeptide can include, in 5’ to 3’ order, nucleotide sequences that encode an HSV-1 gD secretory signal, an UL21 polypeptide or antigenic fragment thereof, a linker, an UL46 polypeptide or antigenic fragment thereof, a linker, an UL47 polypeptide or antigenic fragment thereof, a linker, an UL30.1 polypeptide or antigenic fragment thereof, a linker, an UL40 polypeptide or antigenic fragment thereof, a linker, an UL5.2 polypeptide or antigenic fragment thereof, a linker, an UL5.1 polypeptide or antigenic fragment thereof, a linker, and a MITD.
  • such a polyribonucleotide encodes a polypeptide having an amino acid sequence comprising or consisting of MGGAAARLGAVILFVVIVGLHGVRGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGG SGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFK SGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGGPDA AVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGISCLLYDLSTT ALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVK
  • a polyribonucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity with SEQ ID NO: 692. [0541] In some embodiments, a polyribonucleotide encodes a polypeptide having amino acids 26-853 of amino acid sequence SEQ ID NO: 692.
  • an RNA construct provided herein comprises a polyribonucleotide that encodes one or more GP polypeptides.
  • an RNA construct provided herein comprises a polyribonucleotide that encodes an HSV-2 gC or antigenic portion thereof.
  • an RNA construct provided herein comprises a polyribonucleotide that encodes an HSV-2 gD or antigenic portion thereof.
  • an RNA construct provided herein comprises a polyribonucleotide that encodes an HSV-2 gE or antigenic portion thereof. In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an HSV-2 gB, variant, or antigenic portion thereof. In some embodiments, an RNA construct provided herein comprise a polyribonucleotide that encodes at least one T-cell string polypeptide that comprises one or more HSV T-cell antigens or antigenic portions thereof. [0545] In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gC.
  • an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gD. In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gE. In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gB. In some embodiments, an RNA construct provided herein comprise a polyribonucleotide that encodes at least one T-cell string polypeptide that comprises one or more antigenic portions of HSV T-cell antigens.
  • an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gC and a secretory signal. In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gD and a secretory signal. In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gE and a secretory signal. In some embodiments, an RNA construct provided herein comprises a polyribonucleotide that encodes an antigenic portion of HSV-2 gB and a secretory signal.
  • an RNA construct provided herein comprise a polyribonucleotide that encodes at least one T-cell string polypeptide that comprises one or more antigenic portions of HSV T-cell antigens and a secretory signal.
  • polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5’UTR and/or a 3’ UTR.
  • polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail.
  • polyribonucleotides described herein may comprise a 5’ cap, which may be incorporated during transcription, or joined to a polyribonucleotide post- transcription.
  • 5' Cap A structural feature of RNAs is cap structure at five-prime end (5’).
  • Natural eukaryotic RNA comprises a 7-methylguanosine cap linked to the RNA via a 5 ⁇ to 5 ⁇ -triphosphate bridge resulting in cap0 structure (m7GpppN).
  • RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511–7526, the entire contents of each of which is hereby incorporated by reference.
  • a 5’-cap structure which may be suitable in the context of the present invention is a cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (“anti-reverse cap analogue”), modified ARCA (e.g.
  • RNA e.g., mRNA
  • 5'-cap refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')).
  • a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose.
  • a guanosine nucleoside included in a 5’ cap comprises a 3’O methylation at a ribose (3’OMeG).
  • a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G).
  • a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (m7(3’OMeG)).
  • m7(3’OMeG) a ribose that is notation used in the above paragraph, e.g., “(m 2 7,3’-O )G” or “m7(3’OMeG)”, applies to other structures described herein.
  • providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes.
  • co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator.
  • improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide.
  • alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.
  • a utilized 5’ caps is a cap0, a cap1, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A et al., and Fig.
  • an RNA described herein comprises a cap1 structure. In some embodiments, an RNA described herein comprises a cap2. [0553] In some embodiments, an RNA described herein comprises a cap0 structure. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G).
  • such a cap0 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m 7 )Gppp.
  • a cap0 structure comprises a guanosine nucleoside methylated at the 2’-position of the ribose of guanosine.
  • a cap0 structure comprises a guanosine nucleoside methylated at the 3’-position of the ribose of guanosine.
  • a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m 2 7,2’-O )G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7- position of guanine and at the 2’-position of the ribose ((m 2 7,3’-O )G).
  • a cap1 structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m 7 )G) and optionally methylated at the 2’ or 3’ position pf the ribose, and a 2’O methylated first nucleotide in an RNA ((m 2’-O )N 1 ).
  • a cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3’ position of the ribose, and a 2’O methylated first nucleotide in an RNA 1 ).
  • a cap1 structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2'-O )N 1 ) or (m 2 7,3’-O )Gppp( 2'-O )N 1 ), wherein N 1 is as defined and described herein.
  • a cap1 structure comprises a second nucleotide, N 2 , which is at position 2 and is chosen from A, G, C, or U, e.g., (m 7 )Gppp( 2'-O )N 1 pN 2 or (m 2 7,3’-O )Gppp( 2'-O )N 1 pN 2 , wherein each of N 1 and N 2 is as defined and described herein.
  • a cap2 structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m 7 )G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’O methylated first and second nucleotides in an RNA ((m 2’-O )N 1 p(m 2’-O )N 2 ).
  • a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3’ position of the ribose, and a 2’O methylated first and second nucleotide in an RNA.
  • a cap2 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2'-O )N 1 p( 2'-O )N 2 ) or (m 2 7,3’- O )Gppp( 2'-O )N 1 p( 2'-O )N 2 ), wherein each of N 1 and N 2 is as defined and described herein.
  • the 5’ cap is a dinucleotide cap structure.
  • the 5’ cap is a dinucleotide cap structure comprising N 1 , wherein N 1 is as defined and described herein.
  • the 5’ cap is a dinucleotide cap G*N 1 , wherein N 1 is as defined above and herein, and G* comprises a structure of formula (I): or a salt thereof, wherein each R 2 and R 3 is -OH or -OCH 3 ; and X is O or S.
  • R 2 is -OH.
  • R 2 is -OCH 3 .
  • R 3 is -OH.
  • R 3 is -OCH 3 .
  • R 2 is -OH and R 3 is -OH. In some embodiments, R 2 is -OH and R 3 is -CH 3 . In some embodiments, R 2 is -CH 3 and R 3 is -OH. In some embodiments, R 2 is -CH 3 and R 3 is - CH 3 . [0558] In some embodiments, X is O. In some embodiments, X is S.
  • the 5’ cap is a dinucleotide cap0 structure (e.g., (m 7 )GpppN 1 , (m 2 7,2’- O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1 ), wherein N 1 is as defined and described herein.
  • N 1 is as defined and described herein.
  • the 5’ cap is a dinucleotide cap0 structure (e.g., (m 7 )GpppN 1 , (m 2 7,2’- O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1 ), wherein N 1 is G.
  • the 5’ cap is a dinucleotide cap0 structure (e.g., (m 7 )GpppN 1 , (m 2 7,2’-O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , (m 7 )GppSpN 1 , (m 2 7,2’-O )GppSpN 1 , or (m 2 7,3’-O )GppSpN 1 ), wherein N 1 is A, U, or C.
  • a dinucleotide cap0 structure e.g., (m 7 )GpppN 1 , (m 2 7,2’-O )GpppN 1 , (m 2 7,3’-O )GpppN 1 , wherein N 1 is A, U, or C.
  • the 5’ cap is a dinucleotide cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 , (m 2 7,2’-O )Gppp(m 2’-O )N 1 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 , (m 7 )GppSp(m 2’-O )N 1 , (m 2 7,2’-O )GppSp(m 2’-O )N 1 , or (m 2 7,3’-O )GppSp(m 2’-O )N 1 ), wherein N 1 is as defined and described herein.
  • N 1 is as defined and described herein.
  • the 5’ cap is selected from the group consisting of (m 7 )GpppG (“Ecap0”), (m 7 )Gppp(m 2’-O )G (“Ecap1”), (m 2 7,3’-O )GpppG (“ARCA” or “D1”), and (m 2 7,2’-O )GppSpG (“beta-S-ARCA”).
  • the 5’ cap is (m 7 )GpppG (“Ecap0”), having a structure: or a salt thereof.
  • the 5’ cap is (m 7 )Gppp(m 2’-O )G (“Ecap1”), having a structure: or a salt thereof.
  • the 5’ cap is (m 2 7,3’-O )GpppG (“ARCA” or “D1”), having a structure: or a salt thereof.
  • the 5’ cap is (m2 7,2’-O )GppSpG (“beta-S-ARCA”), having a structure: or a salt thereof.
  • the 5’ cap is a trinucleotide cap structure.
  • the 5’ cap is a trinucleotide cap structure comprising N 1 pN 2 , wherein N 1 and N 2 are as defined and described herein.
  • the 5’ cap is a dinucleotide cap G*N 1 pN 2 , wherein N 1 and N 2 are as defined above and herein, and G* comprises a structure of formula (I): or a salt thereof, wherein R 2 , , .
  • the 5’ cap is a trinucleotide cap0 structure (e.g. (m 7 )GpppN 1 pN 2 , (m 2 7,2’- O )GpppN 1 pN 2 , or (m 2 7,3’-O )GpppN 1 pN 2 ), wherein N 1 and N 2 are as defined and described herein).
  • the 5’ cap is a trinucleotide cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 pN 2 , (m 2 7,2’-O )Gppp(m 2’-O )N 1 pN 2 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 pN 2 ), wherein N 1 and N 2 are as defined and described herein.
  • the 5’ cap is a trinucleotide cap2 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 , (m 2 7,2’-O )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 , (m 2 7,3’- O )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 ), wherein N 1 and N 2 are as defined and described herein.
  • the 5’ cap is selected from the group consisting of (m 2 7,3’-O )Gppp(m 2’-O )ApG (“CleanCap AG”, “CC413”), (m 2 7,3’-O )Gppp(m 2’- O )GpG (“CleanCap GG”), (m 7 )Gppp(m 2’-O )ApG, (m 7 )Gppp(m 2’-O )G, (m 2 7,3’-O )Gppp(m 2 6,2’-O )ApG, and (m 7 )Gppp(m 2’- O )ApU.
  • the 5’ cap is (m 2 7,3’-O )Gppp(m 2’-O )ApG (“CleanCap AG”, “CC413”), having a structure: or a salt there [0566] In some embodiments, the 5’ cap is (m 2 7,3’-O )Gppp(m 2’-O )GpG (“CleanCap GG”), having a structure: or a salt thereo f. [0567] In some embodiments, the 5’ cap is (m 7 )Gppp(m 2’-O )ApG, having a structure:
  • the 5’ cap is (m 7 )Gppp(m 2’-O )GpG, having a structure: or a salt there .
  • the 5’ cap is (m 2 7,3’-O )Gppp(m 2 6,2’-O )ApG, having a structure:
  • the 5’ cap is (m 7 )Gppp(m 2’-O )ApU, having a structure: or a salt thereof.
  • the 5’ cap is a tetranucleotide cap structure.
  • the 5’ cap is a tetranucleotide cap structure comprising N 1 pN 2 pN 3 , wherein N 1 , N 2 , and N 3 are as defined and described herein.
  • the 5’ cap is a tetranucleotide cap G*N 1 pN 2 pN 3 , wherein N 1 , N 2 , and N 3 are as defined above and herein, and G* comprises a structure of formula (I): or a salt thereof, wherein R 2 , [0572]
  • the 5’ cap is a tetranucleotide cap0 structure (e.g.
  • the 5’ cap is a tetranucleotide Cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 pN 2 pN 3 , (m 2 7,2’- O )Gppp(m 2’-O )N 1 pN 2 pN 3 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 pN 2 N 3 ), wherein N 1 , N 2 , and N 3 are as defined and described herein.
  • tetranucleotide Cap1 structure e.g., (m 7 )Gppp(m 2’-O )N 1 pN 2 pN 3 , (m 2 7,2’- O )Gppp(m 2’-O )N 1 pN 2 pN 3 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 pN 2 N 3 ), wherein N 1
  • the 5’ cap is a tetranucleotide Cap2 structure (e.g., (m 7 )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 pN 3 , (m 2 7,2’- O )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 pN 3 , (m 2 7,3’-O )Gppp(m 2’-O )N 1 p(m 2’-O )N 2 pN 3 ), wherein N 1 , N 2 , and N 3 are as defined and described herein.
  • N 1 , N 2 , and N 3 are as defined and described herein.
  • the 5’ cap is selected from the group consisting of (m 2 7,3’-O )Gppp(m 2’- O )Ap(m 2’-O )GpG, (m 2 7,3’-O )Gppp(m 2’-O )Gp(m 2’-O )GpC, (m 7 )Gppp(m 2’-O )Ap(m 2’-O )UpA, and (m 7 )Gppp(m 2’-O )Ap(m 2’-O )GpG.
  • the 5’ cap is (m 2 7,3’-O )Gppp(m 2’-O )Ap(m 2’-O )GpG, having a structure: or a salt thereof.
  • the 5’ cap is (m 2 7,3’-O )Gppp(m 2’-O )Gp(m 2’-O )GpC, having a structure:
  • the 5’ cap is (m 7 )Gppp(m 2’-O )Ap(m 2’-O )UpA, having a structure: or a salt thereo f.
  • the 5’ cap is (m 7 )Gppp(m 2’-O )Ap(m 2’-O )GpG, having a structure:
  • a cap proximal sequence comprises a sequence adjacent to a 5’ cap.
  • a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap structure comprises one or more polynucleotides of a cap proximal sequence.
  • a cap structure comprises an m 7 Guanosine cap and nucleotide +1 (N 1 ) of an RNA polynucleotide.
  • a cap structure comprises an m 7 Guanosine cap and nucleotide +2 (N 2 ) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotides +1 and +2 (N 1 and N 2 ) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotides +1, +2, and +3 (N1, N2, and N3) of an RNA polynucleotide.
  • one or more residues of a cap proximal sequence may be included in an RNA by virtue of having been included in a cap entity (e.g., a cap1 or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase).
  • the 5’ cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises N 1 of the 5’ cap, where N 1 is any nucleotide, e.g., A, C, G or U.
  • the 5’ cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises N 1 and N 2 of the 5’ cap, wherein N 1 and N 2 are independently any nucleotide, e.g., A, C, G or U.
  • the 5’ cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises N 1 , N 2 , and N 3 of the 5’ cap, wherein N 1 , N 2 , and N 3 are any nucleotide, e.g., A, C, G or U.
  • a cap proximal sequence comprises N 1 of a the 5’ cap, and N 2 , N 3 , N 4 and N 5 , wherein N 1 to N 5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap proximal sequence comprises N 1 and N 2 of a the 5’ cap, and N 3 , N 4 and N 5 , wherein N 1 to N 5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap proximal sequence comprises N 1 , N 2 , and N 3 of a the 5’ cap, and N 4 and N 5 , wherein N 1 to N 5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • N 1 is A.
  • N 1 is C.
  • N 1 is G.
  • N 1 is U.
  • N 2 is A.
  • N 2 is C.
  • N 2 is G.
  • N 2 is U.
  • N 3 is A. In some embodiments, N 3 is C. In some embodiments, N 3 is G. In some embodiments, N 3 is U. In some embodiments, N 4 is A. In some embodiments, N 4 is C. In some embodiments, N 4 is G. In some embodiments, N 4 is U. In some embodiments, N 5 is A. In some embodiments, N 5 is C. In some embodiments, N 5 is G. In some embodiments, N 5 is U. It will be understood that each of the embodiments described above and herein (e.g., for N 1 through N 5 ) may be taken singly or in combination and/or may be combined with other embodiments of variables described above and herein (e.g., 5’ caps).
  • a cap proximal sequence comprises A 1 and G 2 of the Cap1 structure, and a sequence comprising: A 3 A 4 U 5 (SEQ ID NO: 150) at positions +3, +4 and +5 respectively of the polyribonucleotide.
  • a nucleic acid e.g., DNA, RNA
  • 5’-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element).
  • a 5’ UTR comprises multiple different sequence elements.
  • the term “untranslated region” or “UTR” is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR).
  • the terms “five prime untranslated region” or “5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide.
  • “5' UTR” refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context.
  • a 5' UTR comprises a Kozak sequence.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap.
  • a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5’ cap.
  • Example 5’ UTRs include a human alpha globin (hAg) 5’UTR or a portion thereof, a TEV 5’ UTR or a portion thereof, a HSP705’ UTR or a portion thereof, or a c-Jun 5’ UTR or a portion thereof.
  • an RNA disclosed herein comprises a hAg 5’ UTR or a portion thereof.
  • an RNA disclosed herein comprises a 5’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5’ UTR with the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 151).
  • an RNA disclosed herein comprises a 5’ UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 151).
  • an RNA disclosed herein comprises a 5’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5’ UTR with the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAACCCGCCACC (SEQ ID NO: 152)(hAg- Kozak/5'UTR).
  • an RNA disclosed herein comprises a 5’ UTR having the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAACCCGCCACC (SEQ ID NO: 152)(hAg-Kozak/5'UTR).
  • a polynucleotide e.g., DNA, RNA
  • a polyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR.
  • poly(A) sequence or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide.
  • Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein.
  • An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical.
  • polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence.
  • polynucleotides disclosed herein comprise interrupted Poly(A) sequence.
  • RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • a poly(A) sequence (SEQ ID NO: 829) of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).
  • a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length.
  • a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly(A) sequence typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1, which is incorporated herein by reference in its entirety, may be used in accordance with the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed.
  • the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U).
  • Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'- end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.
  • the poly(A) sequence (SEQ ID NO: 829) may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides.
  • the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence (SEQ ID NO: 829) may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence (SEQ ID NO: 829) comprises at least 100 nucleotides.
  • the poly(A) sequence (SEQ ID NO: 829) comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.
  • a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less.
  • RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 153).
  • a poly(A) tail comprises a plurality of A residues interrupted by a linker.
  • a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 154).
  • RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 155), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of AAAAAAAAAAAAAAAAAAA
  • a poly(A) tail comprises a plurality of A residues interrupted by a linker.
  • a linker comprises the nucleotide sequence GCAUAUGAC (SEQ ID NO: 156). 5.
  • 3' UTR [0601]
  • an RNA utilized in accordance with the present disclosure comprises a 3'- UTR.
  • the terms “three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a sequence of an RNA molecule that begins following a stop codon of a coding region of an open reading frame sequence.
  • the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.
  • the term “3'- UTR” does preferably not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.
  • an RNA disclosed herein comprises a 3’ UTR comprising an F element d/ I l I b di 3’ UTR i l h i i i i e.
  • element sequence is a 3’-UTR of amino-terminal enhancer of split (AES).
  • an RNA disclosed herein comprises a 3’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATG CTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAG CCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTT GGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 157).
  • an RNA disclosed herein comprises a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATG CTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAG CCTAGCCACACCCCCGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTT GGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 157).
  • an RNA disclosed herein comprises a 3’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3’ UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCA GGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 158).
  • an RNA disclosed herein comprises a 3’ UTR with the sequence of CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCA GGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 158).
  • a 3’UTR is an FI element as described in WO2017/060314, which is herein incorporated by reference in its entirety.
  • RNA Formats At least three distinct formats useful for RNA compositions (e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing RNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying RNA (saRNA). Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end.
  • ORFs open reading frames flanked by untranslated regions
  • an ORF of an uRNA and modRNA vectors encode an antibody agent or portion thereof.
  • An saRNA has multiple ORFs.
  • the RNA described herein may have modified nucleosides.
  • the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.
  • uracil describes one of the nucleobases that can occur in the nucleic acid of RNA.
  • the structure of uracil is: .
  • the structure of uridine is: .
  • UTP uridine 5’-triphosphate
  • Pseudo-UTP Pseudo owing structure: .
  • “Pseudouridine” s o e exa pe o a o e uceos e at is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.
  • Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1 ⁇ ), which has the structure: .
  • N1-methyl-pseudo-UTP has the following structure: .
  • Another exempl m5U which has the structure: .
  • the modified nucleoside is a modified uridine.
  • RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is independently selected from pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), and 5-methyl-uridine (m5U).
  • the modified nucleoside comprises pseudouridine ( ⁇ ).
  • the modified nucleoside comprises N1-methyl-pseudouridine (m1 ⁇ ).
  • the modified nucleoside comprises 5-methyl-uridine (m5U).
  • RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), and 5-methyl-uridine (m5U).
  • the modified nucleosides comprise pseudouridine ( ⁇ ) and N1-methyl-pseudouridine (m1 ⁇ ). In some embodiments, the modified nucleosides comprise pseudouridine ( ⁇ ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1 ⁇ ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), and 5- methyl-uridine (m5U).
  • the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm
  • the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine.
  • modified cytidine in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine.
  • the RNA comprises 5- methylcytidine and one or more selected from pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), and 5-methyl- uridine (m5U).
  • the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1 ⁇ ).
  • the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1 ⁇ ) in place of each uridine.
  • the RNA is “replicon RNA” or simply a “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.”
  • the replicon or self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive- stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856, which is incorporated herein by reference in its entirety).
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a 3’ poly(A) tail.
  • the genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non-structural proteins are typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome.
  • first ORF is larger than the second ORF, the ratio being roughly 2:1.
  • RNA RNA molecule that resembles eukaryotic messenger RNA
  • mRNA messenger RNA
  • the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly- protein (nsP1234).
  • Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA.
  • a second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding an HSV-2 construct described herein.
  • Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system).
  • Trans-replication requires the presence of both these nucleic acid molecules in a given host cell.
  • the nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
  • a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, as well as good tolerability and safety.
  • modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus good tolerability and safety.
  • self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, higher likelihood for efficacy with very low composition (e.g., immunogenic composition, e.g., vaccine) dose.
  • very low composition e.g., immunogenic composition, e.g., vaccine
  • the present disclosure provides particular RNA constructs optimized, for example, for improved manufacturability, encapsulation, expression level (and/or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein. C.
  • Codon Optimization and GC Enrichment refers to alteration of codons in a coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect the typical codon usage of a host organism (e.g., a subject receiving a nucleic acid molecule (e.g., a polyribonucleotide)) without preferably altering the amino acid sequence encoded by the nucleic acid molecule.
  • coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein.
  • codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.”
  • codon- optimization may include increasing guanosine/cytosine (G/C) content of a coding region of RNA described herein as compared to the G/C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.
  • G/C guanosine/cytosine
  • a coding sequence (also referred to as a “coding region”) is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human).
  • a composition e.g., a pharmaceutical composition
  • sequences in such a polynucleotide may differ from wild type sequences encoding the relevant antigen or portion or epitope thereof, even when the amino acid sequence of the antigen or portion or epitope thereof is wild type.
  • strategies for codon optimization for expression in a relevant subject e.g., a human
  • a relevant subject e.g., a human
  • strategies for codon optimization for expression in a particular cell or tissue e.g., a relevant subject
  • a relevant subject e.g., a human
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • codon bias differences in codon usage between organisms
  • codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis.
  • genes may be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are available, for example, at the "Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways.
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as “human codon optimized polynucleotide”).
  • a portion of a polyribonucleotide is codon optimized (e.g., a portion of or the portion encoding a glycoprotein or a portion of or the portion encoding a secretory signal).
  • the entire polyribonucleotide is codon optimized.
  • Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human. Accordingly, in some embodiments, the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 20.
  • the wild type coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with 30 a frequency of 0.10 etc. (see Table 20). Accordingly, in some embodiments, such a procedure ⁇ as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage. Table 20: Human codon usage with frequencies indicated for each amino acid.
  • a coding sequence may be optimized using a multiparametric optimization strategy.
  • optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an RNA level, and/or a translational level.
  • exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); RNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including, e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof.
  • a coding sequence may be optimized by a GeneOptimizer algorithm as described in Fath et al.
  • a coding sequence may be optimized by Eurofins’ adaption and optimization algorithm “GENEius” as described in Eurofins’ Application Notes: Eurofins’ adaption and optimization software “GENEius” in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.
  • a coding sequence utilized in accordance with the present disclosure has G/C content that is increased compared to a coding sequence for an HSV gC, gD, and/or gE (or portion thereof) construct described herein.
  • guanosine/cytidine (G/C) content of a coding region is modified relative to a comparable coding sequence for an HSV gC, gD, and/or gE (or portion thereof) construct described herein, but the amino acid sequence encoded by the polyribonucleotide not modified.
  • G/C guanosine/cytidine
  • GC enrichment may improve translation of a payload sequence.
  • sequences having an increased G (guanosine)/C (cytidine) content are more stable than sequences having an increased A (adenosine)/U (uridine) content.
  • codons which contain A and/or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleosides.
  • G/C content of a coding region of a polyribonucleotide described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild type RNA.
  • G/C content of a coding region of a polyribonucleotide described herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild type RNA.
  • G/C content of a coding region of a polyribonucleotide provided herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild type RNA.
  • G/C content of a coding region of a polyribonucleotide provided herein comprises at the most 65%, such as at the most 64%, such as at the most 63%, such as at the most 62%, such as at the most 61%, such as at the most 60% G/C content compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild type RNA.
  • C content of a coding region of a polyribonucleotide provided herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the C content of the coding region prior to codon optimization, e.g., of the wild type RNA.
  • C content of a coding region of a polyribonucleotide provided herein comprises at the most 40%, such as at the most 39%, such as at the most 38%, such as at the most 37%, such as at the most 36%, such as at the most 35% C content compared to the C content of the coding region prior to codon optimization, e.g., of the wild type RNA.
  • stability and translation efficiency of a polyribonucleotide may incorporate one or more elements established to contribute to stability and/or translation efficiency of the polyribonucleotide; exemplary such elements are described, for example, in PCT/EP2006/009448 incorporated herein by reference.
  • a polyribonucleotide may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, for example so as to increase the GC- content to increase RNA stability and/or to perform a codon optimization and, thus, enhance translation in cells.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 65.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 65, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 70.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 70, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 73.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 73, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 67.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 67, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 68.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 68, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 75.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 75, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 131.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 131, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 132.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 132, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 159.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 159, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 160.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 160, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 161.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 161, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 162.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 162, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 163.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 163, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 164.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 164, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 327.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 327, a 3’ UTR, and a polyA tail.
  • a polyribonucleotide provided herein encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 328.
  • an RNA construct comprises a 5’ cap, a 5’UTR, a polyribonucleotide that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence according to SEQ ID NO: 328, a 3’ UTR, and a polyA tail.
  • RNA Delivery Technologies Provided polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid compositions, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, e.g., Wadhwa et al.
  • one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).
  • lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and/or engineered for systemic delivery of the RNA to target cells.
  • lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject.
  • A. Lipid Compositions 1. Lipids and Lipid-Like Materials [0657] The terms "lipid” and “lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases.
  • One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment.
  • Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • an amphiphilic compound has a polar head attached to a long hydrophobic tail.
  • the polar portion is soluble in water, while the non-polar portion is insoluble in water.
  • the polar portion may have either a formal positive charge, or a formal negative charge.
  • the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt.
  • the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
  • a "lipid-like material” is a substance that is structurally and/or functionally related to a lipid but may not be considered a lipid in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits).
  • lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides.
  • Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol- containing metabolites such as cholesterol.
  • Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur.
  • a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride”.
  • glycosylglycerols which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage.
  • glycerophospholipids usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine.
  • Ceramides are a major subclass of sphingoid base derivatives with an amide-linked fatty acid.
  • the fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups.
  • the glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base.
  • glycosphingolipids such as cerebrosides and gangliosides.
  • Sterols such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains.
  • the minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes. [0669] Lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
  • suitable lipids or lipid-like materials for use in the present disclosure include those described in WO2020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. 2.
  • Cationic or cationically ionizable lipids or lipid-like materials [0671] In some embodiments cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid.
  • cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • Cationic lipids or lipid-like materials are characterized in that they have a net positive charge (e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction.
  • cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
  • a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.
  • cationic lipids include, but are not limited to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3- dimethylammonium propanes; 1,
  • Suitable cationic lipids for use in the present disclosure include those described in WO2020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • Further suitable cationic lipids for use in the present disclosure include those described in WO2010/053572 (including Cl 2-200 described at paragraph [00225]) and WO2012/170930, both of which are incorporated herein by reference for the purposes described herein.
  • Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).
  • formulations that are useful for pharmaceutical compositions can comprise at least one cationic lipid.
  • Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2- DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1
  • amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
  • physiological pH e.g. pH 7.4
  • second pH preferably at or above physiological pH.
  • a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.
  • a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure. 3.
  • formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non- cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials).
  • anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials.
  • optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.
  • a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles.
  • such lipid or lipid-like material is a non-cationic lipid or lipid-like material.
  • a non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • Specific example phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin.
  • Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl
  • a formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.
  • formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.
  • formulations herein include a polymer conjugated lipid such as a pegylated lipid. "Pegylated lipids" comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.
  • a non-cationic lipid in particular a neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation. 4.
  • Lipoplex Particles In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.
  • RNA lipoplex particle contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle. [0692] In certain embodiments, RNA lipoplex particles include both a cationic lipid and an additional lipid. In some embodiments, a cationic lipid is DOTMA and the additional lipid is DOPE.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.
  • RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm.
  • the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm.
  • RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In some embodiments, RNA lipoplex particles have an average diameter of about 400 nm.
  • RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration.
  • the RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase.
  • the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM.
  • Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC).
  • the at least one cationic lipid comprises 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).
  • DOPE 1,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine
  • the liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine (DOPE).
  • DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane
  • DOPE 1,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine
  • Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen.
  • no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs.
  • RNA accumulation and/or RNA expression in antigen presenting cells such as professional antigen presenting cells in the spleen occurs.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells.
  • the antigen presenting cells are dendritic cells and/or macrophages.
  • LNPs Lipid Nanoparticles
  • nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs).
  • LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
  • an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
  • an LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanoparticle.
  • a neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM.
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the neutral lipid is DSPC.
  • a sterol is cholesterol.
  • a polymer conjugated lipid is a pegylated lipid.
  • a pegylated lipid has the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R 12 and R 13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • R 12 and R 13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R 12 and R 13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.
  • the lipid has one of the following structures (IIIA) or (IIIB): ( ) ( ) wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R 6 is, at each occurrence, independently H, OH or C 1 -C 24 alkyl; and n is an integer ranging from 1 to 15. [0706] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures (IIIC) or (IIID): wherein y and z are each independently integers ranging from 1 to 12.
  • the lipid has one of the following structures (IIIE) or (IIIF): .
  • the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ): ; .
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H. In other of the foregoing embodiments, R 6 is C 1 -C 24 alkyl. In other embodiments, R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C 1 -C 24 alkylene or linear C 1 -C 24 alkenylene.
  • R 1 or R 2 is C 6 -C 24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: , wherein: R 7a and R 7b are, at each occurrence, independently H or C 1 -C 12 alkyl; and a is an integer from 2 to 12, and wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • at least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is C 1 -C 8 alkyl.
  • C 1 -C 8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in in Table 21 below.
  • Table 21 Example Compounds of Formula (III). No. Structure No. Structure No. Structure No. Structure No. Structure No. Structure
  • a cationic lipid has one of the structures set forth in Table 22 below.
  • Table 22 Example Cationic Lipid Structures No. Structure No. Structure H O O N [0721] In so e e o e s, a co p ses a cao c p a s a o za e lipid-like material (lipidoid).
  • a cationic lipid has the following structure: [0722] , p p v v g .g., ameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm.
  • lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm.
  • lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm.
  • lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean 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.
  • average size e.g., mean diameter
  • average diameter refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys.57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference).
  • average diameter “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.
  • lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less.
  • lipid nanoparticles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.
  • the “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).
  • Lipid nanoparticles described herein can be characterized by an “N/P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N/P) in RNA.
  • N/P ratio is the molar ratio of cationic (nitrogen) groups (the “N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N/P) in RNA.
  • N/P ratio is the molar ratio of cationic (nitrogen) groups (the “N” in N/P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N/P) in RNA.
  • N + cationic form
  • Use of a single number in an N/P ratio e.g., an N/P ratio of about 5 is intended to refer to that number over 1, e.g., an N/P
  • a lipid nanoparticle described herein has an N/P ratio greater than or equal to 5. In some embodiments, a lipid nanoparticle described herein has an N/P ratio that is about 5, 6, 7, 8, 9, or 10. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 50. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 70. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 120. B.
  • Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos.
  • cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre-determined molar ratio (e.g., ones described herein).
  • lipid nanoparticles are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg/mL in acetate buffer.
  • a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, neutral lipids, and polymer-conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides (e.g., ones described herein).
  • lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps.
  • the flow rates of a lipid solution and a RNA solution into a mixing unit are maintained at a ratio of 1:3.
  • RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.
  • RNA-encapsulated lipid nanoparticles can be processed through filtration.
  • compositions e.g., pharmaceutical compositions comprising one or more polyribonucleotides described herein.
  • an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • USP United States Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • International Pharmacopoeia International Pharmacopoeia
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
  • Pharmaceutical compositions described herein can be administered by appropriate methods known in the art.
  • compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion.
  • parenteral administration which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion.
  • pharmaceutical compositions described herein are formulated for intravenous, intramuscular, or subcutaneous administration.
  • compositions described herein are formulated for intramuscular administration.
  • pharmaceutical compositions described herein are formulated for intravenous administration.
  • pharmaceutically acceptable excipients that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • an agent that delays absorption for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and/or microfiltration.
  • pharmaceutical compositions can be prepared as described herein and/or methods known in the art.
  • a pharmaceutical composition includes ALC-0315; ALC-0159; DSPC; Cholesterol; Sucrose; NaCl; KCl; Na 2 HPO 4 ; KH 2 PO 4 ; Water for injection.
  • normal saline is used as diluent.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to
  • Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and/or method described herein.
  • Relative amounts of polyribonucleotides encapsulated in lipid nanoparticles, a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.
  • pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • active ingredients e.g., polyribonucleotides encapsulated in lipid nanoparticles
  • dosage levels of the active ingredients may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. [0747] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • active ingredients e.g., polyribonucleotides encapsulated in lipid nanoparticles
  • a pharmaceutical composition is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver a dose of about 5 mg RNA/kg.
  • a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions.
  • a pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and/or calcium salts.
  • a pharmaceutical composition provided herein is a preservative-free, sterile RNA-lipid nanoparticle dispersion in an aqueous buffer for intravenous or intramuscular administration.
  • compositions suitable for administration to humans are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. A.
  • compositions comprising a plurality of polyribonucleotides, wherein the plurality of polyribonucleotides comprises a first set of polyribonucleotides that encode one or more glycoprotein (GP) polypeptides as described herein.
  • a combination comprises one polyribonucleotide that encodes a (GP) polypeptide.
  • a combination comprises two or more polyribonucleotides that encode one or more (GP) polypeptides.
  • a combination comprises three or more polyribonucleotides that encode three or more (GP) polypeptides.
  • a combination comprises four or more polyribonucleotides that encode four or more (GP) polypeptides. In some embodiments, a combination comprises three or more polyribonucleotides that encode three or more (GP) polypeptides and a polyribonucleotide that encodes a T-cell string polypeptide. [0753] Also provided herein are combinations that comprise two or more pharmaceutical compositions, wherein each pharmaceutical composition comprises one or more polyribonucleotides as described herein. Also provided herein are combinations that comprise three or more pharmaceutical compositions, wherein each pharmaceutical composition comprises one or more polyribonucleotides as described herein. [0754] Provided herein are combinations that comprise two or more RNA constructs as described herein.
  • a combination comprises three or more RNA constructs as described herein. In some embodiments, a combination comprises four or more RNA constructs as described herein. In some embodiments, a combination comprises five or more RNA constructs as described herein. [0755] Also provided herein are combinations that comprise two or more pharmaceutical compositions, wherein each pharmaceutical composition comprises one or more RNA constructs as described herein. Also provided herein are combinations that comprise three or more pharmaceutical compositions, wherein each pharmaceutical composition comprises one or more RNA constructs as described herein. Also provided herein are combinations that comprise four or more pharmaceutical compositions, wherein each pharmaceutical composition comprises one or more RNA constructs as described herein.
  • a combination comprises a plurality of polyribonucleotides comprising a first set of polyribonucleotides that comprises a polyribonucleotide encoding a GP polypeptide comprising an HSV-2 gC or antigenic portion thereof, and a polyribonucleotide encodes a GP polypeptide comprising an HSV-2 gD or antigenic portion thereof.
  • a combination comprises a plurality of polyribonucleotides comprising a first set of polyribonucleotides that comprises a polyribonucleotide encoding a GP polypeptide comprising an antigenic portion of HSV-2 gC, and a polyribonucleotide encoding a GP polypeptide comprising an antigenic portion of HSV-2 gD.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gC comprises an amino acid sequence according to SEQ ID NO: 65.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gD comprises an amino acid sequence according to SEQ ID NO: 70.
  • a combination comprises (i) a first pharmaceutical composition comprising a polyribonucleotide that encodes a GP polypeptide comprising an HSV-2 gC or antigenic portion thereof, and (ii) a second pharmaceutical composition comprising a polyribonucleotide that encodes a GP polypeptide comprising an HSV-2 gD antigen or antigenic portion thereof.
  • a combination comprises a plurality of polyribonucleotides comprising a first set of polyribonucleotides that comprises a polyribonucleotide encoding a GP polypeptide comprising an antigenic portion of HSV-2 gC, and a polyribonucleotide encoding a GP polypeptide comprising an antigenic portion of HSV-2 gE.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gC comprises an amino acid sequence according to SEQ ID NO: 65.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gE comprises an amino acid sequence according to SEQ ID NO: 73.
  • a combination comprises (i) a first pharmaceutical composition comprising a polyribonucleotide that encodes a GP polypeptide comprising an HSV-2 gC or antigenic portion thereof, and (ii) a second pharmaceutical composition comprising a polyribonucleotide that encodes a GP polypeptide comprising an HSV-2 gE antigen or antigenic portion thereof.
  • a combination comprises (i) a first pharmaceutical composition comprising a first polyribonucleotide that encodes a GP polypeptide comprising an antigenic portion of HSV-2 gC, and (ii) a second pharmaceutical composition comprising a polyribonucleotide that encodes a GP comprising an antigenic portion of HSV-2 gE.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gC comprises an amino acid sequence according to SEQ ID NO: 65.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gE comprises an amino acid sequence according to SEQ ID NO: 73.
  • a combination comprises a plurality of polyribonucleotides comprising a first set of polyribonucleotides that comprises a polyribonucleotide encoding a GP polypeptide comprising an HSV-2 gD or antigenic portion thereof, and a polyribonucleotide encodes a GP polypeptide comprising an HSV-2 gE or antigenic portion thereof.
  • a combination comprises a plurality of polyribonucleotides comprising a first set of polyribonucleotides that comprises a polyribonucleotide encoding a GP polypeptide comprising an antigenic portion of HSV-2 gD, and a polyribonucleotide encoding a GP polypeptide comprising an antigenic portion of HSV-2 gE.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gD comprises an amino acid sequence according to SEQ ID NO: 70.
  • a GP polypeptide comprising an antigenic portion of HSV-2 gE comprises an amino acid sequence according to SEQ ID NO: 73.
  • a combination comprises (i) a first pharmaceutical composition comprising a polyribonucleotide that encodes a GP polypeptide comprising an HSV-2 gD or antigenic portion thereof, and (ii) a second pharmaceutical composition comprising a polyribonucleotide that encodes a GP polypeptide comprising an HSV-2 gE antigen or antigenic portion thereof.
  • a GP polypeptide that comprises an amino acid sequence according to SEQ ID NO: 65 is encoded by a ribonucleic acid sequence according to 106.
  • a GP polypeptide that comprises an amino acid sequence according to SEQ ID NO: 70 is encoded by a ribonucleic acid sequence according to 118.
  • a GP polypeptide that comprises an amino acid sequence according to SEQ ID NO: 73 is encoded by a ribonucleic acid sequence according to 123.
  • technologies of the present disclosure are used in the treatment and/or prophylactic of an HSV infection.
  • Prophylactic purposes of the present disclosure comprise pre-exposure prophylaxis and/or post-exposure prophylaxis.
  • technologies of the present disclosure are used in the treatment and/or prophylaxis of a disorder related to such an HSV (e.g., HSV-1 and/or HSV-2) infection.
  • a disordered related to such an HSV (e.g., HSV-1 and/or HSV-2) infection comprises, for example, a typical symptom and/or a complication of an HSV (e.g., HSV-1 and/or HSV-2) infection.
  • compositions may be useful to detect and/or characterize one or more features of an anti-HSV (e.g., anti-HSV-1 and/or anti-HSV-2) immune response (e.g., by detecting binding to a provided antigen by serum from an infected subject).
  • an anti-HSV e.g., anti-HSV-1 and/or anti-HSV-2
  • an immune response e.g., by detecting binding to a provided antigen by serum from an infected subject.
  • compositions e.g., that are or comprise one or more GP polypeptides and/or one or more T-cell string polypeptides
  • the present disclosure provides use of encoding nucleic acids (e.g., DNA or RNA) to produce encoded antigens and/or use of DNA constructs to produce RNA.
  • technologies of the present disclosure are utilized in a non-limited subject population; in some embodiments, technologies of the present disclosure are utilized in particular subject populations.
  • a subject population comprises an adult population.
  • an adult population comprises subjects between the ages of about 18 years and about 55 years of age (e.g., about 19, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, or 55,).
  • a subject population comprises an elderly population.
  • an elderly population comprises subjects of, about 56 years of age, about 60 years of age, about 70 years of age, or older (e.g., about 60, 65, 70, 75, 80, 85, 90, 95, or 100 years of age).
  • a subject has a weight of at least about 50 kg.
  • a subject has a weight of at least about 51 kg (e.g., about 52, 53, 54, 55, 56,57, 58, 59, 60 kg).
  • a subject has a body mass index (BMI) in a range of about 17.5 kg/m 2 to about 37 kg/m 2 , such as about 18 kg/m 2 to about 36 kg/m 2 , such as about 18.5 kg/m 2 to about 35 kg/m 2 .
  • BMI body mass index
  • a subject has a BMI of at least 17 kg/m 2 , such as at least 17.5 kg/m 2 , such as at least 18 kg/m 2 , such as at least 18.5 kg/m 2 .
  • a subject has a BMI of at the most 40 kg/m 2 , such as at the most 39 kg/m 2 , such as at the most 38 kg/m 2 , such as at the most 37 kg/m 2 , such as at the most 36 kg/m 2 , such as at the most 35 kg/m 2 .
  • a subject population comprises a pediatric population. In some embodiments, a pediatric population comprises subjects approximately 18 years old or younger. In some such embodiments, a pediatric population comprises subjects between the ages of about 1 year and about 18 years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age).
  • a subject population comprises a newborn population.
  • a newborn population comprises subjects about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 months or younger).
  • subject populations to be treated with technologies described herein include infants (e.g., about 12 months or younger) whose mothers did not receive such technologies described herein during pregnancy.
  • subject populations to be treated with technologies described herein may include pregnant women; in some embodiments, infants whose mothers were treated with disclosed technologies during pregnancy (e.g., who received at least one dose, or alternatively only who received both doses), are not vaccinated during the first weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth.
  • infants whose mothers were treated with disclosed technologies during pregnancy e.g., who received at least one dose, or alternatively only who received both doses
  • are not vaccinated during the first weeks, months, or even years e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more post-birth.
  • a subject population is or comprises children aged 6 weeks to up to 17 months of age.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • another pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • therapeutic intervention e.g., to treat or prevent an HSV infection, or another disease, disorder, or condition.
  • a provided pharmaceutical composition may be administered with a protein vaccine, a DNA vaccine, an RNA vaccine, a cellular vaccine, a conjugate vaccine, etc.
  • one or more doses of a provided pharmaceutical composition may be administered together with (e.g., in a single visit) another composition (e.g., vaccine) or other therapy.
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • HSV e.g., HSV-1 and/or HSV-2
  • a provided pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a subject has no prior history of known or suspected herpes simplex vaccination prior to administration of one or more doses of a composition as disclosed herein.
  • a subject does not have febrile illness prior to administration of one or more doses of a composition as disclosed herein. In some embodiments, a subject does not have febrile illness about 72 hours, about 48 hours, about 36 hours, about 24 hours, or about 12 hours prior to administration of one or more doses of a composition as disclosed herein. [0786] In some embodiments, a subject does not have an acute illness prior to administration of one or more doses of a composition as disclosed herein. In some embodiments, a subject does not have an acute illness about 72 hours, about 48 hours, about 36 hours, about 24 hours, or about 12 hours prior to administration of one or more doses of a composition as disclosed herein.
  • a subject has not received a vaccine 0 to 300 days, 0 to 290 days, 0 to 280 days, 0 to 270 days, 0 to 260 days, 0 to 250 days, 0 to 240 days, 0 to 230 days, 0 to 220 days, 0 to 210, 0 to 200 days, 0 to 190 days, 0 to 180 days, 0 to 170 days, 0 to 160 days, 0 to 150 days, 0 to 140 days, 0 to 130 days, 0 to 120 days, 0 to 110 days, 0 to 100 days, 0 to 90 days, 0 to 80 days, 0 to 70 days, 0 to 60 days, 0 to 50 days, 0 to 40 days, 0 to 35 days, 0 to 30 days, 0 to 29 days, 0 to 28 days before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a subject has not received a vaccine about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 60 days, about 70 days, about 80 days, about 90 days, about 100 days, about 125 days, about 150 days, about 175 days, about 190 days, about 200 days, about 210 days, or about 210 days before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a vaccine is not a seasonal influenza vaccine or a medically indicated vaccine.
  • a subject does not receive a vaccine at least 2 weeks to 35 weeks, at least 3 weeks to 34 weeks, or at least 4 weeks to 33 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a subject does not receive a vaccine at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, or at least about 30 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • the vaccine is not a seasonal influenza vaccine or a medically indicated vaccine.
  • a subject has not received blood, plasma products, or immunoglobulins about 0 to 600 days, about 0 to 590 days, about 0 to 580 days, about 0 to 570 days, about 0 to 560 days, about 0 to 550 days, or about 0 to 545 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received an allergy treatment 8 to 45 days, 12 to 40 days, 16 to 38 days, 21 to 35 days, 23 to 32 days, 25 to 30 days, or 26 to 29 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received an allergy treatment about 14 days, about 16 days, about 18 days, about 20 days, about 22 days, about 24 days, about 26 days, about 28 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • an allergy treatment comprises antigen injections.
  • a subject has not received an immunosuppressive medication 7 to 56 days, 14 to 56 days, 21 to 56 days, 28 to 56 days, 35 to 56 days, 42 to 56 days, 49 to 56 days, 7 to 49 days, 14 to 49 days, 21 to 49 days, 28 to 49 days, 35 to 49 days, 42 to 49 days, 7 to 42 days, 14 to 42 days, 21 to 42 days, 28 to 42 days, 35 to 42 days, 7 to 35 days, 14 to 35 days, 21 to 35 days, 28 to 35 days, 7 to 28 days, 14 to 28 days, 21 to 28 days, 7 to 21 days, 14 to 21 days, or 7 to 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received an immunosuppressive medication about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received an immunosuppressive medication about 28 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • an immunosuppressive medication comprises a systemic corticosteroid or radiotherapy.
  • a systemic coriticosteroid is selected from, but not limited to, methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, aldosterone, cloprednol, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, fludrocortisone, flunisolide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, halcinon
  • a coriticosteroid is prednisone.
  • a subject has not received a prophylactic antipyretic and/or an analgesic medication 0 to 600 days, 0 to 550 days, 0 to 500 days, 0 to 500 days, 0 to 450 days, 0 to 400 days, 0 to 350 days, 0 to 300 days, 0 to 250 days, 0 to 200 days, 0 to 150 days, 0 to 150 days, 0 to 150 days, or 0 to 50 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a prophylactic antipyretic medication is selected from, but not limited to, acetaminophen, a non-steroidal anti-inflammatory drug (NSAID), salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, fatelamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen,
  • NSAID non-ster
  • a prophylactic analgesic medication is selected from, but not limited to, acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, fatelamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprof
  • technologies of the present disclosure may be administered to subjects according to a particular dosing regimen.
  • a dosing regimen may involve a single administration; in some embodiments, a dosing regimen may comprise one or more “booster” administrations after the initial administration.
  • initial and boost doses are the same amount; in some embodiments they differ.
  • two or more booster doses are administered.
  • a plurality of doses are administered at regular intervals. In some embodiments, periods of time between doses become longer.
  • one or more subsequent doses is administered if a particular clinical (e.g., reduction in neutralizing antibody levels) or situational (e.g., local development of a new strain) even arises or is detected.
  • administered pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • RNA constructs that encode HSV-2 gC, gD and/or gE constructs are administered in RNA doses of from about 0.1 ⁇ g to about 300 ⁇ g, about 0.5 ⁇ g to about 200 ⁇ g, or about 1 ⁇ g to about 100 ⁇ g, such as about 1 ⁇ g, about 3 ⁇ g, about 10 ⁇ g, about 30 ⁇ g, about 50 ⁇ g, or about 100 ⁇ g.
  • a plurality of booster doses are administered within 6 months of the first dose, or within 12 months of the first dose.
  • 3 doses or fewer are required to achieve effective vaccination (e.g., greater than 60%, and in some embodiments greater than about 70%, about 75%, about 80%, about 85%, about 90% or more) reduction in risk of infection, or of serious disease.
  • not more than two doses are required.
  • a single dose is sufficient.
  • an RNA dose is about 60 ⁇ g or lower, 50 ⁇ g or lower, 40 ⁇ g or lower, 30 ⁇ g or lower, 20 ⁇ g or lower, 10 ⁇ g or lower, 5 ⁇ g or lower, 2.5 ⁇ g or lower, or 1 ⁇ g or lower. In some embodiments, an RNA dose is about 0.25 ⁇ g, at least 0.5 ⁇ g, at least 1 ⁇ g, at least 2 ⁇ g, at least 3 ⁇ g, at least 4 ⁇ g, at least 5 ⁇ g, at least 10 ⁇ g, at least 20 ⁇ g, at least 30 ⁇ g, or at least 40 ⁇ g.
  • an RNA dose is about 0.25 ⁇ g to 60 ⁇ g, 0.5 ⁇ g to 55 ⁇ g, 1 ⁇ g to 50 ⁇ g, 5 ⁇ g to 40 ⁇ g, or 10 ⁇ g to 30 ⁇ g may be administered per dose. In some embodiments, an RNA dose is about 30 ⁇ g. In some embodiments, at least two such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose. In some embodiments, a first booster dose is administered about one month after an initial dose. In some such embodiments, at least one further booster is administered at one-month interval(s).
  • polyribonucleotides can be produced by methods known in the art.
  • polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template.
  • a plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.
  • a DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP).
  • an appropriate RNA polymerase e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase
  • ribonucleotide triphosphates e.g., ATP, CTP, GTP, UTP.
  • polyribonucleotides e.g., ones described herein
  • pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), or 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).
  • pseudouridine ( ⁇ ) can be used to replace uridine triphosphate (UTP).
  • N1-methyl-pseudouridine (m1 ⁇ ) can be used to replace uridine triphosphate (UTP).
  • 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).
  • an RNA polymerase typically traverses at least a portion of a single-stranded DNA template in the 3' ⁇ 5' direction to produce a single-stranded complementary RNA in the 5' ⁇ 3' direction.
  • a polyribonucleotide comprises a polyA tail
  • a polyA tail may be encoded in a DNA template, e.g., by using an appropriately tailed PCR primer, or it can be added to a polyribonucleotide after in vitro transcription, e.g., by enzymatic treatment (e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase).
  • enzymatic treatment e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase.
  • Suitable poly(A) tails are described herein above.
  • a poly(A) tail comprises a nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 153).
  • a poly(A) tail comprises a plurality of A residues interrupted by a linker.
  • a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 154).
  • RNA e.g., mRNA
  • a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life.
  • capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system such as, e.g., capping enzymes of vaccinia virus).
  • a cap may be introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into a polyribonucleotide during transcription (also known as co-transcriptional capping).
  • a GTP fed-batch procedure with multiple additions in the course of the reaction may be used to maintain a low concentration of GTP in order to effectively cap the RNA.
  • Suitable 5' cap are described herein above.
  • a 5' cap comprises m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.
  • digestion can be achieved with the use of DNase I under appropriate conditions.
  • solution for example, in a buffer such as HEPES, a phosphate buffer solution, a citrate buffer solution, an acetate buffer solution; in some embodiments, such solution may be buffered to a pH within a range of, for example, about 6.5 to about 7.5; in some embodiments approximately 7.0.
  • production of polyribonucleotides may further include one or more of the following steps: purification, mixing, filtration, and/or filling.
  • polyribonucleotides can be purified (e.g., in some embodiments after in vitro transcription reaction), for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA portions, and/or or nucleotides.
  • Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure. Certain purification steps may be or include, for example, one or more of precipitation, column chromatography (including, e.g., but not limited to anionic, cationic, hydrophobic interaction chromatography (HIC)), solid substrate-based purification (e.g., magnetic bead-based purification).
  • polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, polyribonucleotides may be purified using hydrophobic interaction chromatography (HIC) and/or diafiltration. In some embodiments, polyribonucleotides may be purified using HIC followed by diafiltration.
  • HIC hydrophobic interaction chromatography
  • dsRNA may be obtained as side product during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination.
  • cellulose materials may be used to remove dsRNA contamination, for examples in some embodiments in a chromatographic format.
  • cellulose materials e.g., microcrystalline cellulose
  • cellulose materials may be used to purify polyribonucleotides according to methods described in WO 2017/182524, the entire content of which is incorporated herein by reference.
  • a batch of polyribonucleotides may be further processed by one or more steps of filtration and/or concentration.
  • polyribonucleotide(s) for example, after removal of dsRNA contamination, may be further subject to diafiltration (e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of polyribonucleotides to a desirable RNA concentration and/or to exchange buffer to a drug substance buffer.
  • diafiltration e.g., in some embodiments by tangential flow filtration
  • polyribonucleotides may be processed through 0.2 ⁇ m filtration before they are filled into appropriate containers.
  • polyribonucleotides and compositions thereof may be manufactured in accordance with a process as described herein, or as otherwise known in the art.
  • polyribonucleotides and compositions thereof may be manufactured at a large scale.
  • a batch of polyribonucleotides can be manufactured at a scale of greater than 1 g, greater than 2 g, greater than 3 g, greater than 4 g, greater than 5 g, greater than 6 g, greater than 7 g, greater than 8 g, greater than 9 g, greater than 10 g, greater than 15 g, greater than 20 g, or higher.
  • RNA quality control may be performed and/or monitored at any time during production process of polyribonucleotides and/or compositions comprising the same.
  • RNA quality control parameters including one or more of RNA identity (e.g., sequence, length, and/or RNA natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or certain steps of a polyribonucleotide manufacturing process, e.g., after in vitro transcription, and/or each purification step.
  • the stability of polyribonucleotides can be assessed under various test storage conditions, for example, at room temperatures vs. fridge or sub-zero temperatures over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer).
  • polyribonucleotides e.g., ones described herein
  • compositions thereof may be stored stable at a fridge temperature (e.g., about 4qC to about 10qC) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer.
  • polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at a sub-zero temperature (e.g., -20qC or below) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer.
  • polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at room temperature (e.g., at about 25°C) for at least 1 month or longer.
  • one or more assessments may be utilized during manufacture, or other preparation or use of polyribonucleotides (e.g., as a release test).
  • one or more quality control parameters may be assessed to determine whether polyribonucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for distribution).
  • quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and/or residual dsRNA. Certain methods for assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for RNA quality assessment.
  • a batch of polyribonucleotides may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of polyribonucleotides can be designated for one or more further steps of manufacturing and/or formulation and/or distribution if RNA quality assessment indicates that such a batch of polyribonucleotides meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of polyribonucleotides does not meet or exceed the acceptance criteria.
  • a batch of polyribonucleotides that satisfy assessment results can be utilized for one or more further steps of manufacturing and/or formulation and/or distribution.
  • IX. DNA Constructs [0820] Among other things, the present disclosure provides DNA constructs, for example that may encode one or more antibody agents as described herein, or components thereof. In some embodiments, DNA constructs provided by and/or utilized in accordance with the present disclosure are comprised in a vector.
  • Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC).
  • a vector is an expression vector.
  • a vector is a cloning vector.
  • a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.).
  • Expression vectors which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest.
  • a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).
  • Cloning vectors are generally used to modify, engineer, and/or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process).
  • a cloning vector may lack expression signals.
  • a vector may include replication elements such as primer binding site(s) and/or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and/or guide RNA binding sites, etc.
  • a vector is a viral vector (e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.
  • a viral vector e.g., an AAV vector
  • a vector is a non-viral vector.
  • a vector is a plasmid.
  • polynucleotide(s) of the present disclosure are included in a DNA construct (e.g., a vector) amenable to transcription and/or translation.
  • an expression vector comprises a polynucleotide that encodes proteins and/or polypeptides of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.).
  • a sequence or sequences that control expression are selected to achieve a desired level of expression.
  • more than one sequence that controls expression are utilized.
  • more than one sequence that controls expression are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality of proteins and/or polypeptides.
  • a plurality of recombinant proteins and/or polypeptides are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic).
  • a plurality of polypeptides are expressed, each of which is expressed from a separate vector.
  • an expression vector comprising a polynucleotide of the present disclosure is used to produce an RNA and/or protein and/or polypeptide in a host cell.
  • a host cell may be in vitro (e.g., a cell line) – for example a cell or cell line (e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for producing polynucleotides of the present disclosure and proteins and/or polypeptides encoded by said polynucleotides.
  • HEK cells Human Embryonic Kidney
  • Chinese Hamster Ovary cells etc.
  • an expression vector is an RNA expression vector.
  • an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix.
  • an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription.
  • a polynucleotide template is generated through PCR as a linear polynucleotide template.
  • a linearized polynucleotide is mixed with enzymes suitable for RNA synthesis, RNA capping and/or purification.
  • the resulting RNA is suitable for producing proteins encoded by the RNA.
  • a vector may be introduced into host cells using transfection.
  • transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine- mediated transfection.
  • a vector may be introduced into a host cell using transduction.
  • transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides.
  • a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer.
  • Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected.
  • growth conditions e.g., temperature, carbon-dioxide levels, growth medium
  • a skilled artisan would recognize culture conditions for host cells selected are well known in the art.
  • X. Dosage Regimens [0833]
  • the present disclosure provides a method of treating or preventing herpes simplex virus (HSV) infection comprising administering to a subject in need thereof a therapeutically effective amount of a composition disclosed herein, in a treatment cycle comprising one or more doses (e.g., one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, or ten doses) of the composition.
  • doses e.g., one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses,
  • a treatment cycle comprises two or more doses (e.g., two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, or ten doses) of the composition.
  • a treatment cycle comprises two doses.
  • a first dose is a priming dose of a composition disclosed herein.
  • a second dose is a booster dose of a composition disclosed herein.
  • a subject is administered one or more doses of a composition disclosed herein prior to infection with HSV (e.g., HSV-1, HSV-2, or a combination thereof).
  • a subject is administered two or more doses of a composition disclosed herein prior to infection with HSV (e.g., HSV-1, HSV-2, or a combination thereof). In some embodiments, a subject is administered three or more doses of a composition disclosed herein prior to infection with HSV (e.g., HSV-1, HSV-2, or a combination thereof).
  • a second dose of the therapeutically effective amount of a composition disclosed herein is administered to a subject 1 day to 24 weeks, 3.5 days to 24 weeks, 1 week to 24 weeks, 2 weeks to 24 weeks, 4 weeks to 24 weeks, 6 weeks to 24 weeks, 8 weeks to 24 weeks, 10 weeks to 24 weeks, 12 weeks to 24 weeks, 16 weeks to 24 weeks, 20 weeks to 24 weeks, 1 day to 20 weeks, 3.5 days to 20 weeks, 1 week to 20 weeks, 2 weeks to 20 weeks, 4 weeks to 20 weeks, 6 weeks to 20 weeks, 8 weeks to 20 weeks, 10 weeks to 20 weeks, 12 weeks to 20 weeks, 16 weeks to 20 weeks, 1 day to 16 weeks, 3.5 days to 16 weeks, 1 week to 16 weeks, 2 weeks to 16 weeks, 4 weeks to 16 weeks, 6 weeks to 16 weeks, 8 weeks to 16 weeks, 10 weeks to 16 weeks, 12 weeks to 16 weeks, 1 day to 12 weeks, 3.5 days to 12 weeks, 1 week to 12 weeks, 2 weeks to 12 weeks, 4 weeks to 12 weeks, 6 weeks to 12 weeks, 8 weeks to 16 weeks, 10 weeks to 16 weeks
  • a second dose of the therapeutically effective amount of a composition disclosed herein is administered 1 week to 14 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject. In some embodiments, a second dose of the therapeutically effective amount of a composition disclosed herein is administered 4 weeks to 12 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject. In some embodiments, a second dose of the therapeutically effective amount of a composition disclosed herein is administered 6 weeks to 10 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • a second dose of the therapeutically effective amount of a composition disclosed herein is administered about 1 week, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, about 20 weeks, or about 24 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject. In some embodiments, a second dose of the therapeutically effective amount of a composition disclosed herein is administered about 8 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • a second dose of the therapeutically effective amount of a composition disclosed herein is administered about 1 day, about 7 days, about 14 days, about 28 days, about 35 days, about 40 days, about 45 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 58 days, about 59 days, or about 60 days after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • a third dose of the therapeutically effective amount of a composition disclosed herein is administered to a subject 1 day to 24 weeks, 3.5 days to 24 weeks, 1 week to 24 weeks, 2 weeks to 24 weeks, 4 weeks to 24 weeks, 6 weeks to 24 weeks, 8 weeks to 24 weeks, 10 weeks to 24 weeks, 12 weeks to 24 weeks, 16 weeks to 24 weeks, 20 weeks to 24 weeks, 1 day to 20 weeks, 3.5 days to 20 weeks, 1 week to 20 weeks, 2 weeks to 20 weeks, 4 weeks to 20 weeks, 6 weeks to 20 weeks, 8 weeks to 20 weeks, 10 weeks to 20 weeks, 12 weeks to 20 weeks, 16 weeks to 20 weeks, 1 day to 16 weeks, 3.5 days to 16 weeks, 1 week to 16 weeks, 2 weeks to 16 weeks, 4 weeks to 16 weeks, 6 weeks to 16 weeks, 8 weeks to 16 weeks, 10 weeks to 16 weeks, 12 weeks to 16 weeks, 1 day to 12 weeks, 3.5 days to 12 weeks, 1 week to 12 weeks, 2 weeks to 12 weeks, 4 weeks to 12 weeks, 6 weeks to 12 weeks, 8 weeks to 16 weeks, 10 weeks to 16 weeks
  • a third dose of the therapeutically effective amount of a composition disclosed herein is administered 4 weeks to 24 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject. In some embodiments, a third dose of the therapeutically effective amount of a composition disclosed herein is administered 12 weeks to 20 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject. In some embodiments, a third dose of the therapeutically effective amount of a composition disclosed herein is administered 14 weeks to 18 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • a third dose of the therapeutically effective amount of a composition disclosed herein is administered about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, or about 24 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • a third dose of the therapeutically effective amount of a composition disclosed herein is administered about 8 weeks after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • a third dose of the therapeutically effective amount of a composition disclosed herein is administered about 10 days, about 20 days, about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, about 90 days, about 100 days, about 105 days, about 110 days, about 111 days, about 112 days, about 113 days, about 114 days, about 115 days, about 116 days, or about 117 days after administration of the first dose of the therapeutically effective amount of the composition to the subject.
  • each of the one or more doses of a therapeutically effective amount of a composition disclosed herein is administered to a subject intramuscularly, subcutaneously, orally, or intranasally.
  • each of the one or more doses of a therapeutically effective amount of a composition disclosed herein is administered to a subject intramuscularly.
  • each of the one or more doses comprises 0.1 to 500 ⁇ g, 0.2 to 500 ⁇ g, 0.5 to 500 ⁇ g, 0.75 to 500 ⁇ g, 1 to 500 ⁇ g.
  • each of the one or more doses comprises 1 ⁇ g to 250 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof.
  • each of the one or more doses comprises 2 ⁇ g to 200 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof. In some embodiments, each of the one or more doses comprises 3 ⁇ g to 100 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof. In some embodiments, each of the one or more doses comprises 3 ⁇ g to 60 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof.
  • each of the one or more doses comprises 3 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof. In some embodiments, each of the one or more doses comprises 10 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof. In some embodiments, each of the one or more doses comprises 30 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof. In some embodiments, each of the one or more doses comprises 60 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof.
  • each of the one or more doses comprises 0.5 ⁇ g, 0.75 ⁇ g, 1.0 ⁇ g, 1.25 ⁇ g, 1.33 ⁇ g, 1.5 ⁇ g, 1.75 ⁇ g, 2.0 ⁇ g, 2.25 ⁇ g, 2.5 ⁇ g, 5 ⁇ g, 7.5 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, or 25 ⁇ g of one or more polyribonucleotides encoding one or more HSV glycoproteins or antigenic fragments thereof.
  • each of the one or more doses comprises 1 ⁇ g of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different HSV glycoprotein or antigenic fragment thereof.
  • each of the one or more doses comprises 3.33 ⁇ g of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different HSV glycoprotein or antigenic fragment thereof.
  • each of the one or more doses comprises 10 ⁇ g of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different HSV glycoprotein or antigenic fragment thereof.
  • each of the one or more doses comprises 20 ⁇ g of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different HSV glycoprotein or antigenic fragment thereof.
  • the present disclosure provides a method of treating or preventing HSV infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein as part of a combination therapy.
  • the present disclosure provides a method of treating or preventing HSV infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein concomitantly with administration of an antipyretic medication.
  • an antipyretic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • an antipyretic medication is administered concurrently with administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • an antipyretic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes before administration of one of more doses of a therapeutically effective amount of a composition as disclosed herein.
  • the antipyretic comprises acetaminophen, a non-steroidal anti-inflammatory drug (NSAID), salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, fatelamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexi
  • NSAID non-steroidal anti-
  • the antipyretic is an NSAID. In certain embodiments, the antipyretic is acetaminophen.
  • the present disclosure provides a method of treating or preventing HSV (HSV-1, HSV-2, or a combination thereof) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein concomitantly with administration of an analgesic medication. In some embodiments, an analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • an analgesic medication is administered concurrently with administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, an analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes before administration of one of more doses of a therapeutically effective amount of a composition as disclosed herein.
  • an analgesic comprises acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, fatelamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprof
  • the analgesic is acetaminophen.
  • the present disclosure provides a method of treating or preventing HSV (HSV-1, HSV-2, or a combination thereof) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein concomitantly with administration of an antipyretic medication and an analgesic medication.
  • an antipyretic medication and analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • an antipyretic medication and analgesic medication is administered concurrently with administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, an antipyretic medication and analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes before administration of one of more doses of a therapeutically effective amount of a composition as disclosed herein. [0851] In some embodiments, an antipyretic medication and/or analgesic medication is acetaminophen.
  • acetaminophen is administered to the subject at a dose of 0.1 g/day to 20 g/day, 0.25 g/day to 20 g/day, 0.5 g/day to 20 g/day, 0.75 g/day to 20 g/day, 1.0 g/day to 20 g/day, 1.25 g/day to 20 g/day, 1.5 g/day to 20 g/day, 1.75 g/day to 20 g/day, 2.0 g/day to 20 g/day, 2.25 g/day to 201 g/day, 2.5 g/day to 20 g/day, 2.75 g/day to 20 g/day, 3.0 g/day to 20 g/day, 3.25 g/day to 20 g/day, 3.5 g/day to 20 g/day, 3.75 g/day to 20 g/day, 4.0 g/day to 20 g/day, 4.25 g/day to 20 g/day,
  • acetaminophen is administered to the subject at a dose of 0.5 g/day to 10 g/day. In some embodiments, acetaminophen is administered to the subject at a dose of 1 g/day to 5 g/day. [0852] In some embodiments, acetaminophen is administered to the subject at a dose of about 0.5 g/day, about 1 g/day, about, about 1.5 g/day, about 2 g/day, about 2.5 g/day, about 3 g/day, about 3.5 g/day, about 4 g/day, about 4.5 g/day, or about 5 g/day.
  • acetaminophen is administered to the subject at a dose of about 4 g/day.
  • the present disclosure provides a method of treating or preventing HSV (HSV-1, HSV-2, or a combination thereof) infection.
  • the present disclosure provides a method of treating or preventing HSV (HSV-1, HSV-2, or a combination thereof) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein, wherein the subject is further administered a medically indicated vaccine at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days before administration of the therapeutically acceptable amount of the composition.
  • a medically indicated vaccine at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 17 days, at least 18 days
  • the subject is further administered a medically indicated vaccine at least 14 days before the administration of the therapeutically acceptable amount of the composition.
  • the present disclosure provides a method of treating or preventing HSV (HSV-1, HSV-2, or a combination thereof) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein, wherein the subject is further administered a medically indicated vaccine at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days after administration of the therapeutically acceptable amount of the composition.
  • a medically indicated vaccine includes, but is not limited to, a rabies vaccine, a tetanus vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a measles mumps rubella (MMR) vaccine, a polio vaccine, a diphtheria vaccine, a varicella vaccine, a pertussis vaccine, a shingles vaccine, a pneumococcal vaccine, a human papillomavirus vaccine (HPV), a meningococcal vaccine, or a rotavirus vaccine.
  • MMR measles mumps rubella
  • HPV human papillomavirus vaccine
  • a medically indicated vaccine is a rabies vaccine or a tetanus vaccine.
  • XII. Monitoring Efficacy [0857]
  • the present disclosure provides a method of treating or preventing HSV (HSV-1, HSV-2, or a combination thereof) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein, wherein the method further comprises collecting one or more samples from the subject after administration of the one or more doses of a therapeutically effective amount of the composition.
  • At least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen samples are collected from the subject after administration of the one or more doses of a therapeutically effective amount of the composition.
  • the one or more samples collected from the subject is a blood volume draw.
  • a sample is collected from the subject about 120 minutes, about 90 minutes, about 60 minutes, about 45 minutes, about 30 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 7.5 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes or about 1 minute before administration of a first dose of one or more doses of a therapeutically effective amount of the composition.
  • a sample is collected from the subject about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 7.5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, or about 120 minutes after administration of a first dose of one or more doses of a therapeutically effective amount of the composition.
  • a sample is collected about 1 week after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 2 weeks after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 4 weeks after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 5 weeks after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 6 weeks after administration of a first dose of one or more doses of a therapeutically effective amount of the composition.
  • a sample is collected about 8 weeks after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 16 weeks after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 7 months after administration of a first dose of one or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 13 months after administration of a first dose of one or more doses of a therapeutically effective amount of the composition.
  • a sample is collected from the subject about 120 minutes, about 90 minutes, about 60 minutes, about 45 minutes, about 30 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 7.5 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes or about 1 minute before administration of a second dose of two or more doses of a therapeutically effective amount of the composition.
  • a sample is collected from the subject about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 7.5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, or about 120 minutes after administration of a second dose of two or more doses of a therapeutically effective amount of the composition.
  • a sample is collected about 1 week after administration of a second dose of two or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 2 weeks after administration of a second dose of two or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 1 month after administration of a second dose of two or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 3 months after administration of a second dose of two or more doses of a therapeutically effective amount of the composition. In some embodiments, a sample is collected about 6 months after administration of a second dose of two or more doses of a therapeutically effective amount of the composition.
  • methods of the present disclosure further comprise a step of measuring levels of HSV (HSV-1 and/or HSV-2) virus-specific neutralizing antibodies in one or more samples collected from the subject.
  • levels of HSV (HSV-1 and/or HSV-2) virus-specific neutralizing antibodies are measured using any one of a number of assays known to persons of ordinary skill in the art.
  • levels of HSV (HSV-1 and/or HSV-2) virus-specific neutralizing antibodies are measured using a plaque reduction neutralization test (PRNT).
  • PRNT plaque reduction neutralization test
  • levels of HSV (HSV-1 and/or HSV-2) virus-specific neutralizing antibodies are measured using a pseudo-viral neutralization test.
  • methods of the present disclosure further comprise a step of measuring levels of neutralizing antibodies in one or more samples collected from a subject, where the neutralizing antibodies are specific for one or more HSV (HSV-1 and/or HSV-2) antigens or antigenic fragments thereof encoded by one or more polyribonucleotides in a composition disclosed herein.
  • levels of neutralizing antibodies are measured using any one of a number of assays known to persons of ordinary skill in the art.
  • ELISA enzyme-linked immunosorbent assay
  • exemplary LNP formulations that are useful for compositions can comprise at least one ionizable aminolipid.
  • exemplary LNP formulations that are useful for compositions can further comprise a helper lipid, which in some embodiments may be or comprise a neutral helper lipid.
  • exemplary LNP formulations that are useful for compositions can further comprise a polymer-conjugated lipid, for example in some embodiments PEG-conjugated lipids.
  • exemplary LNP formulations that are useful for compositions (e.g., immunogenic compositions, e.g., vaccines) as described herein can comprise at least one ionizable aminolipid, at least one helper lipid (e.g., a neutral helper lipid, which in some embodiments may comprise a phospholipid, a steroid, or combinations thereof), and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid).
  • an exemplary LNP formulation may comprise an ionizable aminolipid, a phospholipid, a steroid, and a PEG-conjugated lipid.
  • an ionizable aminolipid may be present in an exemplary LNP formulation within a range of 45 to 55 mol percent, 40 to 50 mol percent, 41 to 49 mol percent, 41 to 48 mol percent, 42 to 48 mol percent, 43 to 48 mol percent, 44 to 48 mol percent of total lipids.
  • an ionizable aminolipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (also known as 6- [N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate).
  • an ionizable aminolipid is or comprises SM-102 (heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate) or an aminolipid as described in Sabnis et al. “ A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates” Mol. Ther. (2018) 26:1509-1519.
  • an ionizable aminolipid is or comprises an ionizable aminolipid as disclosed in US2020/0163878 or WO2018/078053, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • a phospholipid may be present in an exemplary LNP formulation within a range of 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent of total lipids.
  • a phospholipid is or comprises 1,2-Distearoyl-sn-glycero- 3-phosphocholine (DSPC).
  • a sterol may be present in an exemplary LNP formulation within a range of 30 to 50 mol percent, 35 to 45 mol percent or 38 to 43 mol percent of total lipids.
  • a sterol is or comprises cholesterol.
  • a polymer conjugated lipid e.g., PEG-conjugated lipid
  • PEG-conjugated lipid may be present in an exemplary LNP formulation within a range of 1 to 10 mol percent, 1 to 5 mol percent, or 1 to 2.5 mol percent of total lipids.
  • a PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (also known as 2-[2-( ⁇ -methoxy (polyethyleneglycol2000) ethoxy]-N,N- ditetradecylacetamide).
  • a phospholipid is or comprises PEG2000-DMG (1- monomethoxypolyethyleneglycol-2,3- dimyristylglycerol with polyethylene glycol of average molecular weight 2000).
  • a PEG-conjugated lipid is or comprises a PEG-lipid as disclosed in US2020/0163878 or WO2018/078053, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • an exemplary LNP formulation comprises (i) an ionizable aminolipid within a range of 45 to 55 mol percent of total lipids; (ii) a phospholipid within a range of 8 to 12 mol percent of total lipids; (iii) a steroid within a range of 35 to 45 mol percent of total lipids; and (iv) a polymer conjugated (e.g., PEG- conjugated polymer) within a range of 1 to 2 mol percent of total lipids; and RNA molecules as described herein that are encapsulated within or associated with the lipid nanoparticles.
  • a polymer conjugated e.g., PEG- conjugated polymer
  • an exemplary LNP formulation comprises (i) ionizable amino lipid within a range of 45 to 55 mol percent of total lipids; (ii) DSPC within a range of 5 to 15 mol percent of total lipids; (iii) cholesterol within a range of 35 to 45 mol percent of total lipids; and (iv) a PEG-conjugated lipid within a range of 1 to 2 mol percent of total lipids; and RNA molecules as described herein that are encapsulated within or associated with the lipid nanoparticles.
  • an exemplary LNP formulation comprises (i) an ionizable aminolipid within a range of 40 to 50 mol percent of total lipids; (ii) a phospholipid within a range of 5 to 15 mol percent of total lipids; (iii) a steroid within a range of 35 to 45 mol percent of total lipids; and (iv) a polymer conjugated (e.g., PEG- conjugated polymer) within a range of 1 to 10 mol percent of total lipids; and RNA molecules as described herein that are encapsulated within or associated with the lipid nanoparticles.
  • a polymer conjugated e.g., PEG- conjugated polymer
  • an ionizable aminolipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (also known as 6- [N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate).
  • a phospholipid is or comprises 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • a steroid is or comprises cholesterol.

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Abstract

La présente divulgation concerne des compositions pharmaceutiques pour l'administration d'antigènes du HSV (par exemple, un vaccin contre le HSV) et des technologies associées (par exemple, des composants de ceux-ci et/ou des procédés associés).
PCT/US2024/040874 2023-08-03 2024-08-02 Compositions pharmaceutiques pour l'administration d'antigènes du virus de l'herpès simplex et procédés associés Pending WO2025030165A1 (fr)

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