Nucleic acid vaccines for varicella zoster virus (VZV)

Abstract

To provide nucleic acid vaccines.SOLUTION: Vaccines include at least one RNA polynucleotides having an open reading frame encoding at least one varicella zoster virus (VZV) antigen. Methods for preparing and using such vaccines are also described.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 245,234, filed October 22, 2015, U.S. Provisional Application No. 62 / 247,697, filed October 28, 2015, U.S. Provisional Application No. 62 / 335,348, filed May 12, 2016, and U.S. Provisional Application No. 62 / 245,031, filed October 22, 2015, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Chickenpox is an acute infectious disease caused by the varicella-zoster virus (VZV). Varicella-zoster virus is one of eight herpesviruses known to infect humans and vertebrates. VZV is also known as chickenpox virus, varicella virus, shingles virus, and human herpesvirus type 3 (HHV-3). VZV affects humans exclusively, typically causing chickenpox in children, teenagers, and young adults and herpes zoster (shingles) in adults (rarely in children). Primary VZV infection, which results in chickenpox (varicella), can also lead to complications, including viral or secondary bacterial pneumonia. Even after the clinical symptoms of chickenpox have resolved, VZV remains dormant in the nervous system of infected individuals in the trigeminal and dorsal root ganglia (viral latency). In approximately 10–20% of cases, VZV becomes activated later in life and returns to the skin from sensory ganglia, where it causes the disease (rash) known as shingles or herpes zoster. VZV can also cause several neurological conditions ranging from aseptic meningitis to encephalitis. Other serious complications of VZV infection include postherpetic neuralgia, Mollaret's meningitis, multiple herpes zoster, thrombocytopenia, myocarditis, arthritis, and inflammation of arteries in the brain that can lead to stroke, myelitis, ophthalmic zoster, or septic zoster. In rare cases, VZV affects the geniculate ganglion and subsequently causes lesions in certain branches of the facial nerve. Symptoms may include painful blisters on the tongue and ear, unilateral facial weakness, and hearing loss.

[0003] Chickenpox cases have declined by 97% since 1995, primarily due to vaccination. However, an estimated 500,000 to 1 million cases of herpes zoster (shingles) occur each year in the United States alone. The lifetime risk of herpes zoster is estimated to be at least 32%, with aging and cell-mediated immune suppression being the most important risk factors. In fact, it is estimated that 50% of people who survive to age 85 will develop herpes zoster.

[0004] A live-attenuated VZV Oka strain vaccine is available and is marketed in the United States under the trade name VARIVAX® (Merck). A similar, but not identical, VZV vaccine is marketed worldwide as VARILRIX® (GlaxoSmithKline). Since its approval in 1995, it has been included in the recommended childhood vaccination schedule in Australia, the United States, and several other countries. In 2007, the Advisory Committee on Immunization Practices (ACIP) recommended a second dose of the vaccine before school entry to ensure a high level of varicella immunity. From 2001 to 2005, outbreaks were reported in schools with high varicella vaccination rates, demonstrating that a single-dose vaccine policy failed to prevent varicella outbreaks, even in settings where the majority of children were vaccinated and vaccination was performed as expected. Consequently, a two-dose vaccination protocol was adopted; however, breakthrough varicella cases have been reported despite two doses of the vaccine. Furthermore, varicella vaccination has raised concerns that vaccine-induced immunity may not be lifelong and that adults may be more susceptible to more severe disease as immunity from childhood vaccination wanes.

[0005] In 2005, the FDA approved the combined live-attenuated rubella-mumps-rubella-varicella (MMRV) vaccine, PROQUAD™ (Merck), for use in people 12 months to 12 years of age. The attenuated rubella-mumps-rubella vaccine viruses in MMRV are the same as those in the MMR vaccine and are of comparable potency, but the potency of Oka / Merck VZV is higher in the MMRV vaccine than in the single-antigen varicella vaccine.

[0006] In 2006, the U.S. Food and Drug Administration approved ZOSTAVAX® (Merck) for the prevention of shingles (herpes zoster) in people 60 years of age and older (it is now approved for those 50-59 years of age). ZOSTAVAX® contains the same Oka / Merck varicella-zoster virus used in the chickenpox and MMRV vaccines, but with a much higher titer (more than 10 times the viral load) than that present in both vaccines, due to a concentrated formulation designed to elicit an immune response in older adults whose immunity to VZV weakens with age.

[0007] Although the chickenpox vaccine has been shown to be safe in healthy individuals, there is evidence that the immunity conferred by the vaccine to VZV infection wanes over time, leaving vaccinated individuals susceptible to more severe forms of shingles. In addition, there have been reports of individuals developing chickenpox or shingles from chickenpox vaccination. The vaccine can establish a latent infection in nerve ganglia that can then reactivate and cause herpes zoster.

[0008] Furthermore, live-attenuated viruses are not suitable for all subjects, including pregnant women and those with moderate or severe acute illness. Chickenpox is also not suitable or approved for immunocompromised patients, including those with leukemia, lymphoma, systemic malignancies, immunodeficiency disorders, or immunosuppressive therapy. Similarly, individuals with moderate or severe cellular immunodeficiency resulting from human immunodeficiency virus (HIV) infection, including those diagnosed with acquired immunodeficiency syndrome (AIDS), should not receive the chickenpox vaccine. Thus, although immunocompromised individuals are at high risk for morbidity and mortality associated with herpes zoster, this population is not eligible for vaccination with live-attenuated vaccines such as ZOSTAVAX®.

[0009] One million cases of herpes zoster occur each year in the United States. An estimated $1 billion is spent annually in the direct medical costs of herpes zoster in the United States, and treatments for herpes zoster are not always effective or available.

[0010] Deoxyribonucleic acid (DNA) vaccination is a technique used to stimulate humoral and cellular immune responses to foreign antigens, such as VZV antigens. Direct injection of engineered DNA (e.g., naked plasmid DNA) into a living host induces direct production of the antigen by a small number of host cells, resulting in a protective immune response. However, potential problems arise with this technique, including the possibility of insertional mutagenesis, which can lead to the activation of oncogenes or the repression of tumor suppressor genes. Summary of the Invention

[0011] Provided herein are ribonucleic acid (RNA) vaccines based on the discovery that RNA (e.g., messenger RNA (mRNA)) can be safely directed into the body's cellular machinery to produce nearly any protein of interest, ranging from naturally occurring proteins to antibodies and other entirely novel protein constructs that may have therapeutic activity inside or outside of cells. The varicella-zoster virus (VZV) RNA vaccines of the present disclosure can be used to induce a balanced immune response, including both cellular and humoral immunity, against VZV without many of the risks associated with attenuated virus vaccination.

[0012] RNA (e.g., mRNA) vaccines can be used in a variety of settings, depending on the prevalence of infectious diseases or the degree or level of unmet medical need. RNA (e.g., mRNA) vaccines can be used to treat and / or prevent various genotypes, strains, and isolates of VZV. RNA (e.g., mRNA) vaccines have superior properties in that they produce greater antibody titers and more rapid responses compared to commonly available antiviral treatments. Without wishing to be bound by theory, RNA vaccines, such as mRNA polynucleotides, are thought to be better designed to generate the appropriate protein conformation upon translation because RNA vaccines utilize natural cellular machinery. Unlike conventional vaccines, which are produced ex vivo and may elicit undesirable cellular responses, RNA (e.g., mRNA) vaccines are delivered to cell lines in a more natural manner.

[0013] Various VZV amino acid sequences encompassed by the present disclosure are set forth in Tables 1-9. The RNA (e.g., mRNA) vaccines provided herein can comprise at least one RNA polynucleotide encoding at least one of the VZV glycoproteins set forth in Table 1, or a fragment, homolog (e.g., having at least 80%, 85%, 90%, 95%, 98%, or 99% identity), or variant or derivative thereof.

[0014] Some embodiments of the present disclosure provide a VZV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide a VZV vaccine comprising at least one RNA polynucleotide having an open reading frame encoding two or more VZV antigenic polypeptides or immunogenic fragments or epitopes thereof. Some embodiments of the present disclosure provide a VZV vaccine comprising two or more RNA polynucleotides having open reading frames encoding two or more VZV antigenic polypeptides or immunogenic fragments or epitopes thereof.

[0015] In some embodiments, the antigenic polypeptide is a VZV glycoprotein. For example, the VZV glycoprotein can be VZV gE, gI, gB, gH, gK, gL, gC, gN, or gM, or an immunogenic fragment or epitope thereof. In some embodiments, the antigenic polypeptide is a VZV gE polypeptide. In some embodiments, the antigenic polypeptide is a VZV gI polypeptide. In some embodiments, the antigenic polypeptide is a VZV gB polypeptide. In some embodiments, the antigenic polypeptide is a VZV gH polypeptide. In some embodiments, the antigenic polypeptide is a VZV gK polypeptide. In some embodiments, the antigenic polypeptide is a VZV gL polypeptide. In some embodiments, the antigenic polypeptide is a VZV gC polypeptide. In some embodiments, the antigenic polypeptide is a VZV gN polypeptide. In some embodiments, the antigenic polypeptide is a VZV gM polypeptide. In some embodiments, the VZV glycoprotein is encoded by the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] In some embodiments, the VZV glycoprotein is a variant gE polypeptide. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide lacking the anchor domain (ER retention domain). In some embodiments, the truncated VZV gE polypeptide comprises (or consists of, or consists essentially of) amino acids 1-561 of a VZV gE polypeptide. In some embodiments, the truncated VZV gE polypeptide comprises (or consists of, or consists essentially of) amino acids 1-561 of SEQ ID NO: 10. In some embodiments, the truncated VZV gE polypeptide comprises (or consists of, or consists essentially of) amino acids 1-573 of SEQ ID NO: 18. In some embodiments, the truncated VZV gE polypeptide comprises (or consists of, or consists essentially of) amino acids 1-573 of SEQ ID NO: 10. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide lacking the carboxy-terminal tail domain. In some embodiments, the truncated VZV gE polypeptide comprises (or consists of, or consists essentially of) amino acids 1-573 of a VZV gE polypeptide. In some embodiments, the truncated VZV gE polypeptide comprises (or consists of, or consists essentially of) amino acids 1-573 of SEQ ID NO:34.

[0017] In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with ER retention, wherein the mutation(s) in the one or more motifs reduce retention of the VZV gE polypeptide in the ER and / or Golgi apparatus. In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with targeting of gE to the Golgi apparatus or trans-Golgi network (TGN), wherein the mutation(s) in the one or more motifs reduce targeting or localization of the VZV gE polypeptide to the Golgi apparatus or TGN. In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with VZV gE internalization or gE endocytosis, wherein the mutation(s) in the one or more motifs reduce endocytosis of the VZV gE polypeptide. In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more phosphorylated acidic motifs, such as SSTT (SEQ ID NO: 122). In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having a Y582G mutation. In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having a Y569A mutation. In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having a Y582G mutation and a Y569A mutation. In some embodiments, the variant VZV gE polypeptide is an antigenic fragment comprising amino acids 1-573 of VZV gE and having a Y569A mutation. In some embodiments, the variant VZV gE polypeptide is an antigenic fragment comprising SEQ ID NO: 38.

[0018] In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having an Igκ sequence. In some embodiments, the variant VZV gE polypeptide is SEQ ID NO: 14. In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having an AEAADA sequence (SEQ ID NO: 58) replacing SESTDT (SEQ ID NO: 59). This replaces the Ser / Thr-rich "SSTT" (SEQ ID NO: 122) acidic cluster with an Ala-rich sequence. In some embodiments, the variant VZV gE polypeptide is SEQ ID NO: 26. In some embodiments, the VZV gE polypeptide has the AEAADA sequence (SEQ ID NO:58), the variant VZV gE polypeptide also has at least one mutation in one or more motifs associated with ER / Golgi retention, TGN localization, or endocytosis (e.g., has a Y582G mutation, a Y569A mutation, or both a Y582G mutation and a Y569A mutation), and / or has at least one mutation in one or more phosphorylated acidic motifs, such as the SSTT (SEQ ID NO:122) motif. In some embodiments, the variant VZV gE polypeptide is or comprises the amino acid sequence of SEQ ID NO:30.

[0019] In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having an additional sequence at the C-terminus that promotes secretion of the polypeptide or its localization to the cell membrane. In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having an Igκ sequence at the C-terminus. In some embodiments, the VZV gE polypeptide has an additional sequence at the C-terminus that promotes secretion (e.g., an Igκ sequence at the C-terminus), and has at least one mutation in one or more motifs associated with ER retention, TGN localization, or endocytosis (e.g., a Y582G mutation, a Y569A mutation, or both a Y582G mutation and a Y569A mutation), and / or has at least one mutation in one or more phosphorylated acidic motifs, such as the SSTT (SEQ ID NO: 122) motif. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide that lacks the anchor domain (ER retention domain) and has an additional sequence at the C-terminus that promotes secretion of the polypeptide (e.g., an Igκ sequence at the C-terminus). In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1-561 and has an Igκ sequence at the C-terminus. In some embodiments, the variant polypeptide is SEQ ID NO: 22. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide that lacks the carboxy-terminal tail domain and has an additional sequence at the C-terminus that promotes secretion of the polypeptide (e.g., an Igκ sequence at the C-terminus). In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1-573 and has an Igκ sequence at the C-terminus.

[0020] In some embodiments, the antigenic polypeptide comprises two or more glycoproteins. In some embodiments, the two or more glycoproteins are encoded by a single RNA polynucleotide. In some embodiments, the two or more glycoproteins are encoded by two or more RNA polynucleotides, e.g., each glycoprotein is encoded by a separate RNA polynucleotide. In some embodiments, the two or more glycoproteins can be any combination of VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of VZV gE and at least one of gI, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of VZV gI and at least one of VZV gE, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of VZV gE, gI, and at least one of gB, gH, gK, gL, gC, gN, and gM polypeptides or immunogenic fragments or epitopes thereof.

[0021] In some embodiments, two or more VZV glycoproteins are gE and gI. In some embodiments, two or more VZV glycoproteins are gE and gB. In some embodiments, two or more VZV glycoproteins are gI and gB. In some embodiments, two or more VZV glycoproteins are gE, gI, and gB. In some embodiments, two or more VZV glycoproteins are gE and gH. In some embodiments, two or more VZV glycoproteins are gI and gH. In some embodiments, two or more VZV glycoproteins are gE, gI, and gH. In some embodiments, two or more VZV glycoproteins are gE and gK. In some embodiments, two or more VZV glycoproteins are gI and gK. In some embodiments, two or more VZV glycoproteins are gE, gI, and gK. In some embodiments, two or more VZV glycoproteins are gE and gL. In some embodiments, two or more VZV glycoproteins are gI and gL. In some embodiments, two or more VZV glycoproteins are gE, gI, and gL. In some embodiments, two or more VZV glycoproteins are gE and gC. In some embodiments, two or more VZV glycoproteins are gI and gC. In some embodiments, two or more VZV glycoproteins are gE, gI, and gC. In some embodiments, two or more VZV glycoproteins are gE and gN. In some embodiments, two or more VZV glycoproteins are gI and gN. In some embodiments, two or more VZV glycoproteins are gE, gI, and gN. In some embodiments, two or more VZV glycoproteins are gE and gM. In some embodiments, two or more VZV glycoproteins are gI and gM. In some embodiments, two or more VZV glycoproteins are gE, gI, and gM.

[0022] In some embodiments, the vaccine comprises any two or more VZV glycoproteins (e.g., any of the variant VZV gEs disclosed in the preceding paragraphs and in the Examples and Figures), where the VZV gE is any of the variant VZV gE glycoproteins disclosed herein, e.g., a variant gE such as any of the variant VZV gEs disclosed in the preceding paragraphs and in the Examples and Figures.

[0023] In some embodiments, the VZV vaccine comprises two or more RNA polynucleotides having open reading frames encoding two or more VZV antigenic polypeptides, or immunogenic fragments or epitopes thereof (either encoded by a single RNA polynucleotide or by two or more RNA polynucleotides (e.g., each glycoprotein is encoded by a separate RNA polynucleotide)), wherein the two or more VZV glycoproteins are a variant gE (e.g., any of the variant gE polypeptides disclosed in the preceding paragraphs herein) and a VZV glycoprotein selected from gI, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more VZV glycoproteins are a variant gE (e.g., any of the variant gE polypeptides disclosed in the preceding paragraphs herein) and gI. In some embodiments, the glycoprotein is VZV gI and a variant VZV gE, and the variant VZV gE polypeptide is a truncated polypeptide lacking the anchor domain (ER retention domain) (e.g., a truncated VZV gE polypeptide comprising amino acids 1-561 of SEQ ID NO: 10). In some embodiments, the glycoprotein is VZV gI and a variant VZV gE, and the variant VZV gE polypeptide is a truncated polypeptide lacking the carboxy-terminal tail domain (e.g., a truncated VZV gE polypeptide comprising amino acids 1-573 of SEQ ID NO: 18). In some embodiments, the glycoprotein is VZV gI and a variant VZV gE, wherein the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with ER retention, TGN localization, and / or endocytosis (e.g., the variant VZV gE has a Y582G mutation, a Y569A mutation, or both the Y582G and Y569A mutations), and / or has at least one mutation in one or more phosphorylated acidic motifs, such as the SSTT (SEQ ID NO: 122) motif.In some embodiments, the glycoprotein is VZV gI or variant VZV gE, and the variant VZV gE polypeptide is an antigenic fragment comprising amino acids 1-573 of VZV gE and having a Y569A mutation. In some embodiments, the glycoprotein is VZV gI or variant VZV gE, and the variant VZV gE polypeptide is a full-length VZV gE polypeptide having the AEAADA (SEQ ID NO: 58) sequence. In some embodiments, the glycoprotein is VZV gI or variant VZV gE, and the VZV gE polypeptide has the AEAADA (SEQ ID NO: 58) sequence and a Y582G mutation, a Y569A mutation, or both the Y582G and Y569A mutations. In some embodiments, the glycoprotein is VZV gI or variant VZV gE, and the VZV gE polypeptide is a full-length VZV gE polypeptide having an additional sequence at the C-terminus that promotes secretion of the polypeptide (e.g., an Igκ sequence). In some embodiments, the glycoprotein is VZV gI or variant VZV gE, and the VZV gE polypeptide is a full-length VZV gE polypeptide having an Igκ sequence and a Y582G mutation, a Y569A mutation, or both the Y582G and Y569A mutations. In some embodiments, the glycoprotein is VZV gI or variant VZV gE, and the VZV gE polypeptide is a truncated VZV gE polypeptide lacking the anchor domain (ER retention domain) and having an Igκ sequence. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide comprising amino acids 1-561 or amino acids 1-573 and having an Igκ sequence at the C-terminus.

[0024] In any of the above-described embodiments, the VZV vaccine may further comprise live attenuated VZV, whole inactivated VZV, or VZV virus-like particles (VLPs). In some embodiments, the live-attenuated VZV, fully inactivated VZV, or VZV VLPs are selected from or derived from the following strains and genotypes: VZV strain E1, genotypes E1_32_5, E1_Kel, E1_Dumas, E1_Russia1999, E1_SD, E1_MSP, E1_36, E1_49, E1_BC, E1_NH29; VZV strain E2, genotypes E2_03-500, E2_2, E2_11, E2_HJO; VZV strain J, genotype pOka; VZV strain M1, genotype M1_CA123; VZV strain M2, genotypes M2_8 and M2_DR; and VZV strain M4, genotypes Spain4242, France4415, and Italy4053.

[0025] Other RNA vaccines containing RNA polynucleotides encoding other viral protein components of VZV (e.g., tegument proteins) are also encompassed by the present disclosure. Accordingly, some embodiments of the present disclosure provide VZV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one VZV tegument protein, or an antigenic fragment or epitope thereof. Some embodiments of the present disclosure provide VZV vaccines comprising at least one RNA polynucleotide having an open reading frame encoding at least one VZV tegument protein, or an immunogenic fragment or epitope thereof, and at least one VZV glycoprotein, or an immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide VZV vaccines comprising at least one RNA polynucleotide having an open reading frame encoding at least one VZV tegument protein, or an immunogenic fragment or epitope thereof, and at least one RNA polynucleotide having an open reading frame encoding at least one VZV tegument protein, or an immunogenic fragment or epitope thereof, and at least one VZV glycoprotein, or an immunogenic fragment or epitope thereof. In some embodiments, the RNA vaccine comprises an RNA (e.g., mRNA) polynucleotide(s) encoding one or more VZV tegument proteins and one or more VZV glycoproteins selected from VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the VZV glycoprotein is a VZV gE polypeptide or an immunogenic fragment or epitope thereof. In some embodiments, the VZV glycoprotein is a VZV gI polypeptide or an immunogenic fragment or epitope thereof. In some embodiments, the VZV glycoprotein is a variant VZV gE polypeptide, such as any of the variant VZV gE polypeptides disclosed herein. In some embodiments, the VZV glycoprotein is a VZV gE glycoprotein or a VZV gI glycoprotein, or an immunogenic fragment or epitope thereof.

[0026] In some embodiments, the at least one RNA polynucleotide is encoded by at least one nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:41, and homologs having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:41, and homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99. ... In some embodiments, at least one RNA polynucleotide is encoded by at least one fragment (e.g., a fragment having an antigenic sequence or at least one epitope) of a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:41, and homologs having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to a nucleic acid sequence selected from SEQ ID NOs:1-8 and 41. In some embodiments, at least one RNA polynucleotide is encoded by at least one epitope of a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:41.

[0027] In some embodiments, at least one RNA polynucleotide is a gE polypeptide encoded by SEQ ID NO: 1. In some embodiments, at least one RNA polynucleotide is a gI polypeptide encoded by SEQ ID NO: 2. In some embodiments, at least one RNA polynucleotide is a truncated gE polypeptide encoded by SEQ ID NO: 3. In some embodiments, at least one RNA polynucleotide is a truncated gE polypeptide encoded by SEQ ID NO: 5. In some embodiments, at least one RNA polynucleotide is a truncated gE polypeptide with a Y569A mutation encoded by SEQ ID NO: 6. In some embodiments, at least one RNA polynucleotide is a gE polypeptide with the AEAADA sequence of SEQ ID NO: 58 encoded by SEQ ID NO: 7. In some embodiments, at least one RNA polynucleotide is a gE polypeptide with a Y582G mutation and the AEAADA sequence (SEQ ID NO: 58) encoded by SEQ ID NO: 8. In some embodiments, at least one RNA polynucleotide is a gE polypeptide encoded by SEQ ID NO: 41.

[0028] In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes an antigenic polypeptide having at least 90% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes an antigenic polypeptide having at least 95% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 96% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes an antigenic polypeptide having at least 97% identity to the amino acid sequence of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes an antigenic polypeptide having at least 98% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes an antigenic polypeptide having at least 99% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55.

[0029] In some embodiments, the open reading frame encoding the VZV polypeptide is codon-optimized. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein selected from any of SEQ ID NOS: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, the at least one RNA polynucleotide comprises an mRNA sequence identified by any one of SEQ ID NOS: 92-108. In some embodiments, the mRNA sequence identified by any one of SEQ ID NOS: 92-108 is codon-optimized to encode an antigenic VZV polypeptide that is as immunogenic as or more immunogenic than the antigenic VZV polypeptide encoded by any one of SEQ ID NOS: 92-108.

[0030] In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 10, wherein the RNA (e.g., mRNA) polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 10, wherein the RNA (e.g., mRNA) polynucleotide has more than 80% identity to the wild-type mRNA sequence, but does not include the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 42, wherein the RNA (e.g., mRNA) polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 42, wherein the RNA (e.g., mRNA) polynucleotide has more than 80% identity to the wild-type mRNA sequence, but does not include the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 14, wherein the RNA (e.g., mRNA) polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 14, wherein the RNA (e.g., mRNA) polynucleotide has more than 80% identity to the wild-type mRNA sequence, but does not include the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 26, wherein the RNA (e.g., mRNA) polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 26, wherein the RNA (e.g., mRNA) polynucleotide has more than 80% identity to the wild-type mRNA sequence, but does not include the wild-type mRNA sequence.In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 30, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide encodes the antigenic protein of SEQ ID NO: 30, wherein the RNA (e.g., mRNA) polynucleotide has more than 80% identity to, but does not include, the wild-type mRNA sequence. In some embodiments, at least one RNA (e.g., mRNA) polynucleotide is encoded by a sequence selected from any of SEQ ID NOs: 1-8 and SEQ ID NO: 41, and comprises at least one chemical modification.

[0031] In some embodiments, the VZV vaccine is multivalent. In some embodiments, the RNA polynucleotide comprises a polynucleotide sequence derived from a VZV E1 strain, e.g., including any one or more of the genotypes E1_32_5, E1_Kel, E1_Dumas, E1_Russia1999, E1_SD, E1_MSP, E1_36, E1_49, E1_BC, and E1_NH29. In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises a polynucleotide sequence derived from a VZV E2 strain, e.g., including any one or more of the genotypes E2_03-500, E2_2, E2_11, and E2_HJO. In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises a polynucleotide sequence derived from a VZV J strain, e.g., including the genotype pOka. In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises a polynucleotide sequence derived from a VZV M1 strain, e.g., including genotype M1_CA123. In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises a polynucleotide sequence derived from a VZV M2 strain, e.g., including genotypes M2_8 and M2_DR. In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises a polynucleotide sequence derived from a VZV M4 strain, e.g., including any one or more of genotypes Spain4242, France4415, and Italy4053.

[0032] Some embodiments of the present disclosure provide a VZV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof and at least one 5'-end cap. In some embodiments, the 5'-end cap is 7mG(5')ppp(5')NlmpNp. Some embodiments of the present disclosure provide a VZV vaccine comprising at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof, wherein the at least one RNA (e.g., mRNA) polynucleotide has at least one chemical modification. In some embodiments, the at least one RNA (e.g., mRNA) polynucleotide further comprises a second chemical modification. In some embodiments, the at least one RNA (e.g., mRNA) polynucleotide having at least one chemical modification has a 5'-end cap. In some embodiments, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, all (100%) of the uridines of at least one RNA polynucleotide comprise a chemical modification, such as an N1-methylpseudouridine modification or an N1-ethylpseudouridine modification.

[0033] Some embodiments of the present disclosure provide a VZV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracils in the open reading frame have a chemical modification, and optionally, the vaccine is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, 100% of the uracils in the open reading frame are modified to include N1-methylpseudouridine.

[0034] Some embodiments of the present disclosure provide a VZV vaccine formulated in cationic lipid nanoparticles. In some embodiments, the cationic lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the cationic lipid is an ionic cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

[0035] In some embodiments, the lipid is [ka] is.

[0036] In some embodiments, the lipid is [ka] is.

[0037] In some embodiments, the cationic lipid nanoparticles have a molar ratio of about 20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid. In some embodiments, the nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the nanoparticles have a neutral net charge at neutral pH. In some embodiments, the nanoparticles have an average diameter of 50-200 nm.

[0038] Some embodiments of the present disclosure provide methods for inducing an antigen-specific immune response in a subject, comprising administering to the subject a VZV RNA (e.g., mRNA) vaccine in an amount effective to produce an antigen-specific immune response. In some embodiments, the antigen-specific immune response comprises a T cell response or a B cell response. In some embodiments, the antigen-specific immune response comprises a T cell response and a B cell response. In some embodiments, the method of producing an antigen-specific immune response involves a single administration of the vaccine. In some embodiments, the method further comprises administering a booster dose of the vaccine to the subject. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0039] Also provided herein is a VZV RNA (e.g., mRNA) vaccine for use in a method of inducing an antigen-specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen-specific immune response.

[0040] Further provided herein is the use of a VZV RNA (e.g., mRNA) vaccine in the manufacture of a medicament for use in a method of inducing an antigen-specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen-specific immune response.

[0041] Some aspects of the present disclosure provide methods for preventing or treating VZV infection, including administering a VZV RNA (e.g., mRNA) vaccine of the present disclosure to a subject. In some embodiments, the VZV RNA (e.g., mRNA) vaccine is formulated in an amount effective to generate an antigen-specific immune response in the subject.

[0042] Some embodiments of the present disclosure provide a method of inducing an antigen-specific immune response in a subject, the method comprising administering to the subject a VZV RNA (e.g., mRNA) vaccine described herein in an amount effective to produce an antigen-specific immune response in the subject.

[0043] In some embodiments, the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is increased by at least 1 log compared to a control, hi some embodiments, the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is increased by 1 to 3 logs compared to a control.

[0044] In some embodiments, the titer of anti-VZV antigenic polypeptide antibodies produced in a subject is increased by at least 2-fold compared to a control. In some embodiments, the titer of anti-VZV antigenic polypeptide antibodies produced in a subject is increased by at least 5-fold compared to a control. In some embodiments, the titer of anti-VZV antigenic polypeptide antibodies produced in a subject is increased by at least 10-fold compared to a control. In some embodiments, the titer of anti-VZV antigenic polypeptide antibodies produced in a subject is increased by 2- to 10-fold compared to a control.

[0045] In some embodiments, the control is the titer of anti-VZV antigenic polypeptide antibodies produced in a subject who has not received a VZV vaccine. In some embodiments, the control is the titer of anti-VZV antigenic polypeptide antibodies produced in a subject who has received a live-attenuated VZV vaccine or an inactivated VZV vaccine. In some embodiments, the control is the titer of anti-VZV antigenic polypeptide antibodies produced in a subject who has received a recombinant or purified VZV protein vaccine. In some embodiments, the control is the titer of anti-VZV antigenic polypeptide antibodies produced in a subject who has received a VZV virus-like particle (VLP) vaccine.

[0046] In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant VZV protein vaccine reduced by at least half, and the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-VZV antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant or purified VZV protein vaccine, or a live-attenuated VZV vaccine or an inactivated VZV vaccine, or a VZV VLP vaccine.

[0047] In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant VZV protein vaccine reduced by at least 4-fold, and the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-VZV antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant or purified VZV protein vaccine, or a live-attenuated VZV vaccine or an inactivated VZV vaccine, or a VZV VLP vaccine.

[0048] In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant VZV protein vaccine reduced by at least 10-fold, and the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-VZV antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant or purified VZV protein vaccine, or a live-attenuated VZV vaccine or an inactivated VZV vaccine, or a VZV VLP vaccine.

[0049] In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant VZV protein vaccine reduced by at least 100-fold, and the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-VZV antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant or purified VZV protein vaccine, or a live-attenuated VZV vaccine or an inactivated VZV vaccine, or a VZV VLP vaccine.

[0050] In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant VZV protein vaccine reduced by at least 1 / 1000, and the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-VZV antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant or purified VZV protein vaccine, or a live-attenuated VZV vaccine or an inactivated VZV vaccine, or a VZV VLP vaccine.

[0051] In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant VZV protein vaccine reduced by 1 / 2 to 1 / 1000, and the titer of anti-VZV antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-VZV antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant or purified VZV protein vaccine, or a live-attenuated VZV vaccine or an inactivated VZV vaccine, or a VZV VLP vaccine.

[0052] In some embodiments, the effective amount is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg, or 25 to 200 μg. In some embodiments, the effective amount is a total dose of 50 μg, 100 μg, 200 μg, 400 μg, 800 μg, or 1000 μg. In some embodiments, the effective amount is a total dose of 200 μg. In some embodiments, the effective amount is a total dose of 50 μg to 400 μg. In some embodiments, the effective amount is a total dose of 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, or 400 μg. In some embodiments, the effective amount is a 25 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 50 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 100 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 200 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 400 μg dose administered to the subject a total of two times. In some embodiments, the effective amount is a 500 μg dose administered to the subject a total of two times.

[0053] In some embodiments, the efficacy (or efficacy rate) of a VZV RNA (eg, mRNA) vaccine against VZV is greater than 60%.

[0054] Vaccine efficacy can be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1;201(11):1607-10). For example, vaccine efficacy can be determined by double-blind, randomized, controlled clinical trials. Vaccine efficacy can be expressed as the proportional reduction in incidence rate (AR) between test cohorts of unvaccinated (ARU) and vaccinated (ARV), and can be calculated from the relative risk (RR) of disease in the vaccinated group using the following formula: Efficacy = (ARU-ARV) / ARU x 100, and Efficacy = (1-RR) x 100

[0055] Similarly, vaccine efficacy can be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1;201(11):1607-10). Vaccine efficacy is an assessment of how well a vaccine (which may already be proven highly effective) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under more natural conditions than controlled clinical trials. Vaccine efficacy is proportional to vaccine efficacy (potency), but is influenced by how well the target group in the population is immunized and by other non-vaccine-related factors that affect "real-world" outcomes such as hospitalizations, outpatient visits, or costs. For example, a retrospective case-control analysis can be used to compare vaccination rates between a series of infected cases and appropriate controls. Vaccine efficacy can be expressed as a rate difference using the odds ratio (OR) of developing an infection despite vaccination. Effectiveness rate = (1-OR) x 100

[0056] In some embodiments, the efficacy (or effectiveness rate) of a VZV RNA (e.g., mRNA) vaccine against VZV is greater than 65%. In some embodiments, the efficacy (or effectiveness rate) of a vaccine against VZV is greater than 70%. In some embodiments, the efficacy (or effectiveness rate) of a vaccine against VZV is greater than 75%. In some embodiments, the efficacy (or effectiveness rate) of a vaccine against VZV is greater than 80%. In some embodiments, the efficacy (or effectiveness rate) of a vaccine against VZV is greater than 85%. In some embodiments, the efficacy (or effectiveness rate) of a vaccine against VZV is greater than 90%.

[0057] In some embodiments, the vaccine immunizes a subject against VZV for up to one year (e.g., for one VZV season). In some embodiments, the vaccine immunizes a subject against VZV for up to two years. In some embodiments, the vaccine immunizes a subject against VZV for more than two years. In some embodiments, the vaccine immunizes a subject against VZV for more than three years. In some embodiments, the vaccine immunizes a subject against VZV for more than four years. In some embodiments, the vaccine immunizes a subject against VZV for five to ten years.

[0058] In some embodiments, the subject receiving a VZV RNA (e.g., mRNA) vaccine is between about 12 months and about 10 years of age (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age). In some embodiments, the subject receiving a VZV RNA (e.g., mRNA) vaccine is between about 12 months and about 15 months of age (e.g., about 12, 12.5, 13, 13.5, 14, 14.5, or 15 months of age). In some embodiments, the subject receiving a VZV RNA (e.g., mRNA) vaccine is between about 4 years and about 6 years of age (e.g., about 4, 4.5, 5, 5.6, or 6 years of age).

[0059] In some embodiments, the subject is a young adult between about 20 and about 50 years of age (eg, about 20, 25, 30, 35, 40, 45, or 50 years of age).

[0060] In some embodiments, the subject is an elderly subject about 60 years of age, about 70 years of age or older (eg, about 60, 65, 70, 75, 80, 85, or 90 years of age).

[0061] In some embodiments, the subject has been exposed to, is infected with, or is at risk for infection by VZV.

[0062] In some embodiments, the subject is immunocompromised (has a disorder of the immune system, eg, an immune system disease or an autoimmune disease).

[0063] Some aspects of the present disclosure provide varicella-zoster virus (VZV) RNA (e.g., mRNA) vaccines containing a signal peptide linked to a VZV antigenic polypeptide. Thus, in some embodiments, the VZV RNA (e.g., mRNA) vaccine contains at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding the signal peptide linked to a VZV antigenic peptide. Also provided herein are nucleic acids encoding the VZV RNA (e.g., mRNA) vaccines disclosed herein.

[0064] Another aspect of the present disclosure provides a varicella-zoster virus (VZV) vaccine comprising a signal peptide linked to a VZV antigenic polypeptide. In some embodiments, the VZV antigenic polypeptide is a VZV glycoprotein. In some embodiments, the VZV glycoprotein is selected from VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM. In some embodiments, the VZV glycoprotein is a VZV gE or a variant VZV gE polypeptide.

[0065] In some embodiments, the signal peptide is an IgE signal peptide. In some embodiments, the signal peptide is an IgE HC (Ig heavy chain epsilon-1) signal peptide. In some embodiments, the signal peptide has the sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 56). In some embodiments, the signal peptide is an IgGκ signal peptide. In some embodiments, the signal peptide has the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 57). In some embodiments, the signal peptide is selected from the Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS, SEQ ID NO: 109), the VSV g protein signal sequence (MKCLLYLAFLFIGVNCA, SEQ ID NO: 110), and the Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA, SEQ ID NO: 111).

[0066] Also provided herein are nucleic acids encoding the VZV vaccines disclosed herein. Such VZV vaccines comprise at least one ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding a signal peptide linked to a VZV antigenic polypeptide. In some embodiments, the VZV antigenic peptide is a VZV glycoprotein. In some embodiments, the VZV glycoprotein is selected from VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM. In some embodiments, the VZV antigenic peptide is VZV gE or a variant of a gE polypeptide.

[0067] In some embodiments, an effective amount of a VZV RNA (e.g., mRNA) vaccine (e.g., a single dose of a VZV vaccine) results in a 2-fold to 200-fold (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200-fold) increase in serum neutralizing antibodies to VZV compared to a control. In some embodiments, a single dose of a VZV RNA (e.g., mRNA) vaccine results in an about 5-fold, 50-fold, or 150-fold increase in serum neutralizing antibodies to VZV compared to a control. In some embodiments, a single dose of a VZV RNA (e.g., mRNA) vaccine results in an about 2-fold to 10-fold or about 40-60-fold increase in serum neutralizing antibodies to VZV compared to a control.

[0068] In some embodiments, the efficacy of an RNA vaccine RNA (e.g., mRNA) can be significantly improved when combined with a flagellin adjuvant, particularly when mRNA encoding one or more antigens is combined with mRNA encoding flagellin.

[0069] RNA (e.g., mRNA) vaccines combined with flagellin adjuvants (e.g., mRNA-encoded flagellin adjuvants) have superior properties compared to commercially available vaccine formulations in that they can produce greater antibody titers and more rapid responses. Without wishing to be bound by theory, it is believed that RNA vaccines, such as mRNA polynucleotides, are better designed to generate the appropriate protein conformations for both the antigen and adjuvant upon translation because RNA (e.g., mRNA) vaccines utilize natural cellular machinery. Unlike traditional vaccines, which are produced ex vivo and can elicit undesirable cellular responses, RNA (e.g., mRNA) vaccines are delivered to cell lines in a more natural manner.

[0070] Some embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines comprising at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide or immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide), and at least one RNA (e.g., mRNA polynucleotide) having an open reading frame encoding a flagellin adjuvant.

[0071] In some embodiments, the at least one flagellin polypeptide (e.g., the encoded flagellin polypeptide) is a flagellin protein. In some embodiments, the at least one flagellin polypeptide (e.g., the encoded flagellin polypeptide) is an immunogenic flagellin fragment. In some embodiments, the at least one flagellin polypeptide and the at least one antigenic polypeptide are encoded by a single RNA (e.g., mRNA) polynucleotide. In other embodiments, the at least one flagellin polypeptide and the at least one antigenic polypeptide are each encoded by different RNA polynucleotides.

[0072] In some embodiments, at least one flagellin polypeptide has at least 80%, at least 85%, at least 90%, or at least 95% identity to a flagellin polypeptide having the sequence of any of SEQ ID NOs: 115-117.

[0073] In some embodiments, the nucleic acid vaccines described herein are chemically modified. In other embodiments, the nucleic acid vaccines are unmodified.

[0074] Yet another aspect provides compositions and methods for vaccinating a subject, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic VZV polypeptide, wherein the RNA polynucleotides do not comprise a stabilizing element, and the vaccine is not co-formulated with or co-administered with an adjuvant.

[0075] In another aspect, the present invention provides a composition and method for vaccinating a subject, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first viral antigenic polypeptide, wherein the nucleic acid vaccine is administered to the subject at a dose of 10 μg / kg to 400 μg / kg. In some embodiments, the dose of the RNA polynucleotide is 1 to 5 μg, 5 to 10 μg, 10 to 15 μg, 15 to 20 μg, 10 to 25 μg, 20 to 25 μg, 20 to 50 μg, 30 to 50 μg, 40 to 50 μg, 40 to 60 μg, 60 to 80 μg, 60 to 100 μg, 50 to 100 μg, 80 to 120 μg, 40 to 120 μg, 40 to 150 μg, 50 to 150 μg, 50 to 200 μg, 80 to 200 μg, 10 to 50 μg, 15 to 20 μg, 10 to 50 μg, 15 to 20 μg, 10 to 25 μg, 20 to 50 μg, 30 to 50 μg, 40 to 50 μg, 40 to 60 μg, 60 to 80 μg, 60 to 100 μg, 50 to 100 μg, 80 to 120 μg, 40 to 120 μg, 40 to 150 μg, 50 to 150 μg, 50 to 200 μg, 80 to 200 μg, 10 to 50 μg, 15 to 20 μg, 15 to 20 μg, 15 to The nucleic acid vaccine may be administered intradermally or intramuscularly. In some embodiments, the nucleic acid vaccine is administered to a subject via intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to a subject on day 0. In some embodiments, a second dose of the nucleic acid vaccine is administered to a subject on day 21.

[0076] In some embodiments, the nucleic acid vaccine administered to a subject comprises a 25 μg dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to a subject comprises a 100 μg dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to a subject comprises a 50 μg dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to a subject comprises a 75 μg dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to a subject comprises a 150 μg dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to a subject comprises a 400 μg dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to a subject comprises a 200 μg dose of RNA polynucleotide. In some embodiments, the RNA polynucleotide accumulates at 100-fold higher levels in local lymph nodes compared to distal lymph nodes. In other embodiments, the nucleic acid vaccine is chemically modified; in other embodiments, the nucleic acid vaccine is not chemically modified.

[0077] An aspect of the present invention provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotides do not comprise a stabilizing element and a pharmaceutically acceptable carrier or excipient, and wherein no adjuvant is included in the vaccine. In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence that is GC-enriched relative to a wild-type sequence.

[0078] An aspect of the present invention provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, the RNA polynucleotides being present in a formulation for in vivo administration to a host that confers an antibody titer against the first antigen that exceeds the antibody retention standard at a percentage acceptable to human subjects. In some embodiments, the antibody titer produced by the mRNA vaccine of the present invention is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is greater than that of a protein vaccine. In other embodiments, the neutralizing antibody titer produced by the mRNA vaccine of the present invention is greater than that of an adjuvanted protein vaccine. In still other embodiments, the titer of neutralizing antibodies produced by the mRNA vaccines of the present invention is 1,000 to 10,000, 1,200 to 10,000, 1,400 to 10,000, 1,500 to 10,000, 1,000 to 5,000, 1,000 to 4,000, 1,800 to 10,000, 2,000 to 10,000, 2,000 to 5,000, 2,000 to 3,000, 2,000 to 4,000, 3,000 to 5,000, 3,000 to 4,000, or 2,000 to 2,500. Neutralizing titers are typically expressed as the highest serum dilution required to achieve a 50% reduction in plaque counts.

[0079] Also provided is a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, the RNA polynucleotides being in a formulation for in vivo administration to a host that induces a higher, longer-lasting antibody titer than that elicited by an mRNA vaccine encoding the first antigenic polypeptide formulated with a stabilizing element or adjuvant. In some embodiments, the RNA polynucleotide is formulated to produce neutralizing antibodies within one week after a single administration. In some embodiments, the adjuvant is selected from a cationic peptide and an immunostimulatory nucleic acid. In some embodiments, the cationic peptide is protamine.

[0080] Some embodiments provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame with at least one chemical modification or optionally no chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotides are in a formulation for in vivo administration to a host such that the host's antigen expression level significantly exceeds the antigen expression level achieved by an mRNA vaccine encoding the first antigenic polypeptide formulated with a stabilizing element or adjuvant.

[0081] Another aspect provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame with at least one chemical modification or optionally no chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is at least 10-fold less than the RNA polynucleotide required to produce an equivalent antibody titer with an unmodified mRNA vaccine. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 μg.

[0082] Aspects of the invention also provide unit-of-use vaccines comprising 10 μg to 400 μg of one or more RNA polynucleotides having an open reading frame with at least one chemical modification, or optionally no chemical modification, encoding a first antigenic polypeptide, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

[0083] Aspects of the present invention provide methods for creating, maintaining, or restoring antigenic memory against a VZV strain in an individual or a population of individuals, the methods comprising administering to the individual or population an antigenic memory booster nucleic acid vaccine, the nucleic acid vaccine comprising: (a) at least one RNA polynucleotide, the polynucleotide comprising at least one chemical modification, or optionally no chemical modification, and two or more codon-optimized open reading frames, the open reading frames encoding a set of reference antigenic polypeptides; and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration, and subcutaneous administration. In some embodiments, the administering step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition. In some embodiments, the administering step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition in combination with electroporation.

[0084] An embodiment of the present invention provides a method of vaccinating a subject, the method comprising administering to the subject a single dose of 25 μg / kg to 400 μg / kg of a nucleic acid vaccine comprising an effective amount of one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, to vaccinate the subject.

[0085] Another aspect provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is at least 10-fold less than the RNA polynucleotide required to produce an equivalent antibody titer with an unmodified mRNA vaccine. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 μg.

[0086] Another aspect provides a nucleic acid vaccine comprising an LNP-formulated RNA polynucleotide having an open reading frame that does not contain modified nucleotides (unmodified), the open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is at least 10-fold less than the RNA polynucleotide required to produce an equivalent antibody titer when an unmodified mRNA vaccine that is not formulated with LNP is used. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 μg.

[0087] The data presented in the examples show that the use of the formulation of the present invention significantly improves immune responses. The data demonstrate the effectiveness of both the chemically modified and unmodified RNA vaccines of the present disclosure. Surprisingly, in contrast to the prior art approach that prefers to use unmodified mRNA in vaccine production and formulate it into a carrier, it has been discovered herein that chemically modified mRNA-LNP vaccines require significantly lower effective mRNA doses than unmodified mRNA, i.e., 1 / 10 lower than unmodified mRNA formulated in a carrier other than LNP. Both chemically modified and unmodified RNA vaccines of the present invention produce better immune responses than mRNA vaccines formulated in different lipid carriers.

[0088] In another aspect, the invention encompasses a method for treating an elderly subject 60 years of age or older, comprising administering to the subject an effective amount of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a VZV antigenic polypeptide, thereby vaccinating the subject.

[0089] In another aspect, the invention encompasses a method for treating a juvenile subject 17 years of age or younger, comprising administering to the subject an effective amount of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a VZV antigenic polypeptide, thereby vaccinating the subject.

[0090] In another aspect, the invention encompasses a method of treating an adult subject, comprising administering to the subject an effective amount of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a VZV antigenic polypeptide, thereby vaccinating the subject.

[0091] The RNA polynucleotide is one of SEQ ID NOs: 1-8 and 41 and comprises at least one chemical modification. In other embodiments, the RNA polynucleotide is one of SEQ ID NOs: 1-8 and 41 and does not comprise any nucleotide modification or is unmodified. In still other embodiments, the at least one RNA polynucleotide encodes an antigenic protein of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42 and 45-55 and comprises at least one chemical modification. In other embodiments, the RNA polynucleotide encodes an antigenic protein of any of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42 and 45-55 and does not comprise any nucleotide modification or is unmodified.

[0092] In a preferred embodiment, the vaccines of the present invention (e.g., LNP-encapsulated mRNA vaccines) result in prophylactically and / or therapeutically effective levels, concentrations, and / or titers of antigen-specific antibodies in the blood or serum of vaccinated subjects. As defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in a subject, e.g., a human subject. In an exemplary embodiment, the antibody titer is expressed as the reciprocal of the highest dilution (serial dilution) that still produces a positive result. In an exemplary embodiment, the antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA). In an exemplary embodiment, the antibody titer is determined or measured by a neutralization assay, e.g., a microneutralization assay. In certain embodiments, the antibody titer measurement is expressed as a ratio of 1:40, 1:100, etc.

[0093] In exemplary embodiments of the invention, an effective vaccine produces an antibody titer of greater than 1:40, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, or greater than 1:10000. In exemplary embodiments, this antibody titer is produced or achieved by 10 days post-vaccination, 20 days post-vaccination, 30 days post-vaccination, 40 days post-vaccination, or 50 days post-vaccination or greater. In exemplary embodiments, the titer is produced or achieved after administering a single dose of vaccine to a subject. In other embodiments, the titer is produced or achieved after multiple administrations, e.g., a first dose and a second dose (e.g., a booster dose).

[0094] In exemplary aspects of the invention, antigen-specific antibodies are measured in μg / ml, or in IU / L (International Units / Liter), or mIU / ml (milli-International Units / ml). In exemplary embodiments of the invention, an effective vaccine provides >0.5 μg / ml, >0.1 μg / ml, >0.2 μg / ml, >0.35 μg / ml, >0.5 μg / ml, >1 μg / ml, >2 μg / ml, >5 μg / ml, or >10 μg / ml. In exemplary embodiments of the invention, an effective vaccine provides >10 mIU / ml, >20 mIU / ml, >50 mIU / ml, >100 mIU / ml, >200 mIU / ml, >500 mIU / ml, or >1000 mIU / ml. In exemplary embodiments, the antibody level or concentration is achieved or produced 10 days after vaccination, 20 days after vaccination, 30 days after vaccination, 40 days after vaccination, or 50 days or more after vaccination. In exemplary embodiments, the level or concentration is achieved or produced after administering a single dose of vaccine to the subject. In other embodiments, the level or concentration is achieved or produced after multiple administrations, e.g., a first dose and a second dose (e.g., a booster dose). In exemplary embodiments, the antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, the antibody level or concentration is determined or measured by a neutralization assay, e.g., a microneutralization assay.

[0095] Each of the limitations of the present invention may encompass various embodiments of the present invention. Accordingly, it is contemplated that each of the limitations of the present invention involving any one element or combination of elements may be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways.

[0096] The accompanying drawings are not intended to be drawn to scale. In the drawings, identical or nearly identical components shown in various figures are respectively represented by like numerals. For clarity, not every component may be labeled in every drawing. [Brief explanation of the drawings]

[0097] [Figure 1] FIG. 1 is a schematic diagram showing the proposed viral pathway of varicella zoster. [Figure 2] FIG. 1 is a schematic diagram of a construct encoding VZV gE (Oka strain). [Figure 3] 1 shows the study design and injection schedule for immunization of BALB / C mice with VZV gE antigen encoded by mRNA formulated in MC3. [Figure 4] 1 is a schematic diagram showing various variant VZV gE antigens, which from top to bottom show SEQ ID NOS: 120, 132, and 58, respectively. [Figure 5] 1 is a graph depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of mice vaccinated with various VZV gE mRNAs compared to the VARIVAX® vaccine. [Figure 6] 1 is a graph depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of mice vaccinated with various VZV gE mRNAs compared to the VARIVAX® vaccine. [Figure 7] 1 is a graph depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of mice vaccinated with various VZV gE mRNAs compared to the VARIVAX® vaccine. [Figure 8] 8A and 8B show confocal microscopy images of human melanoma (MeWo) cells stained with an antibody representing the Golgi apparatus. [Figure 9]9A-9C show confocal microscopy images of MeWo cells stained with an antibody to VZV gE to demonstrate expression and transport of VZV gE. The sequence AEAADA shown in FIG. 9C is SEQ ID NO: 58. [Figure 10] 10A and 10B are phylogenetic diagrams depicting various VZV wild-type genotypes. [Figure 11-1] 11A and 11B are graphs depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of mice vaccinated with VZV gE variant mRNA compared to the ZOSTAVAX® vaccine. The sequence AEAADA shown in Figures 11A and 11B is SEQ ID NO:58. [Figure 11-2] This is a continuation of 11-1. [Figure 12] 12A and 12B are graphs depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of mice vaccinated with VZV gE variant mRNA compared to the ZOSTAVAX® vaccine. The sequence AEAADA shown in Figures 12A and 12B is SEQ ID NO:58. [Figure 13] 13 is a graph showing the results of an ELISA assay measuring antibody titers in the serum of mice immunized with a VZV gE variant mRNA vaccine. The anti-VZV gE response in mice induced by the VZV gE variant mRNA is greater than the response induced by ZOSTAVAX®. The gE variant mRNA of the deletion-Y569A from GE-574 induced an immune response that was 1 log greater than that of ZOSTAVAX®. The sequence AEAADA shown in FIG. 13 is SEQ ID NO: 58. [Figure 14] Figure 14A is a graph depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of mice vaccinated with VZV gE variant mRNA (Groups 1-5) or VZV-gE-deleted 574_Y569A (Group 6) after primary exposure with the ZOSTAVAX® vaccine. The sequence AEAADA shown in Figure 14A is SEQ ID NO: 58. Figure 14B is a graph depicting the EC50 determination. [Figure 15] Figures 15A and 15B are graphs depicting the results of T cell analysis of mice vaccinated with VZV gE variant mRNA (Groups 1-5) or VZV-gE-deleted 574_Y569A (Group 6) after primary exposure with the ZOSTAVAX® vaccine. The sequence AEAADA shown in Figures 15A and 15B is SEQ ID NO: 58. [Figure 16-1] Figures 16A and 16B are graphs depicting the results of an ELISA assay showing anti-VZV gE IgG levels in the serum of rhesus macaques vaccinated with either ZOSTAVAX® or ZOSTAVAX after primary exposure with VZV-gE-deleted 574_Y569A or VZV-gE-deleted 574_Y569A. Figure 16C is a graph depicting EC50 and EC10 determinations. Figures 16D, 16E, and 16F are graphs depicting the results of T cell analysis of rhesus macaques vaccinated with either ZOSTAVAX® or ZOSTAVAX® after primary exposure with VZV-gE-deleted 574_Y569A or VZV-gE-deleted 574_Y569A. [Figure 16-2] This is a continuation of Figure 16-1. [Figure 16-3] This is a continuation of Figure 16-2. DETAILED DESCRIPTION OF THE INVENTION

[0098] Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines comprising at least one RNA (e.g., mRNA) polynucleotide encoding a varicella-zoster virus (VZV) antigen. Varicella-zoster virus (VZV) has at least five clades. Clades 1 and 3 include European / North American strains, clade 2 includes Asian strains, particularly from Japan, and clade 5 appears to be centered in India. Clade 4 includes several strains from Europe, but its geographic origin needs further clarification. Phylogenetic analysis of VZV genome sequences has separated wild-type strains into nine genotypes (E1, E2, J, M1, M2, M3, M4, VIII, and IX). Sequence analysis of 342 clinical samples of varicella and varicella zoster from 18 European countries identified the following VZV genotype distribution: E1 221 (65%), E2 87 (25%), M1 20 (6%), M2 3 (1%), and M4 11 (3%). No M3 or J strains were observed. Of 165 clinical isolates of varicella and varicella zoster from Australia and New Zealand, 127 eastern Australian isolates contained 67 E1, 30 E2, 16 J, 10 M1, and 4 M2 genotypes; and 38 New Zealand isolates contained 25 E1, 8 E2, and 5 M1 genotypes.

[0099] VZV is an alphaherpesvirus that exists as a spherical, multilayered structure approximately 200 nm in diameter. The viral genome is surrounded by a capsid protein structure, which is covered by an amorphous layer of tegument protein. These two structures are surrounded by a lipid envelope, each approximately 8 nm long and dotted with multiple viral glycoproteins displayed on the outside of the virion. The lipid envelope encapsulates a 100 nm nucleocapsid, consisting of 162 icosahedron-like hexameric and pentameric capsomeres. The tegument, consisting of virally encoded proteins and enzymes, is located in the space between the nucleocapsid and the viral envelope. The viral envelope is obtained from the host cell membrane and contains virally encoded glycoproteins.

[0100] The VZV genome is a single, linear, double-stranded DNA molecule of 124,884 base pairs with at least 70 open reading frames. The genome has two major isoforms, P (prototype) and IS (inverted S), which exist at equal frequencies, totaling 90–95%, depending on the orientation of the S segment. The L segment can also be inverted, resulting in a total of four linear isoforms (IL and ILS).

[0101] VZV is closely related to herpes simplex virus (HSV) and shares considerable homology with it. The VZV genome is the smallest of the human herpesviruses and encodes at least 71 unique proteins (ORFs 0 to 68), with three additional open reading frames (ORFs 69 to 71, respectively) that repeat the previous open reading frame (ORFs 64 to 62). Only a portion of the encoded proteins form the structure of the virus particle. These proteins include nine glycoproteins: ORFs 5 (gK), 9A (gN), 14 (gC), 31 (gB), 37 (gH), 50 (gM), 60 (gL), 67 (gI), and 68 (gE). The known envelope glycoproteins (gB, gC, gE, gH, gI, gK, gL, gN, and gM) correspond to those of HSV, but there is no counterpart to HSV gD. VZV also does not produce LAT (latency-associated transcript), which plays a key role in establishing HSV latency (herpes simplex virus). The encoded glycoproteins, gE, gI, gB, gH, gK, gL, gC, gN, and gM, function at different stages of the viral replication cycle. The most abundant glycoprotein present in infected cells and mature virions is glycoprotein E (gE, ORF68). It is a major component of the virion envelope and is essential for viral replication. Glycoprotein I (gI, ORF67) forms a complex with gE in infected cells. This complex promotes endocytosis of both glycoproteins and targets them to the trans-Golgi network (TGN), where the final viral envelope is acquired. Glycoprotein I (gI) is required within the TGN for efficient membrane fusion during VZV envelopment and replication. VZV gE and gI have been observed to complex on the surface of infected host cells. Glycoprotein B (ORF31), the second most abundant protein and thought to be involved in viral entry, binds to neutralizing antibodies. Glycoprotein H is thought to have a fusion function that facilitates cell-to-cell spread of the virus.Antibodies to gE, gB, and gH are abundant after natural infection and vaccination and have been shown to neutralize viral activity in vitro.

[0102] Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines comprising at least one polynucleotide encoding at least one VZV antigenic polypeptide. The VZV RNA vaccines provided herein can be used to induce a balanced immune response, including both cellular and humoral immunity, without many of the risks associated with DNA vaccines and live-attenuated vaccines. Various RNA (e.g., mRNA) vaccines disclosed herein have induced immune responses in BALB / C mice, the results of which are discussed in detail in the Examples section. Specifically, RNA (e.g., mRNA) polynucleotide vaccines containing open reading frames encoding one or more of various VZV antigens induced significant immune responses compared to conventional VZV vaccines (e.g., attenuated VZV viruses). VZV RNA (e.g., mRNA) polynucleotide vaccines encoding either VZV gE or variant VZV gE disclosed herein, when administered intramuscularly (IM) or intradermally (ID), induced significant immune responses after two doses. VZV glycoproteins and tegument proteins have been shown to be antigenic. VZV glycoproteins, fragments thereof, and epitopes thereof are encompassed by the present disclosure.

[0103] The entire contents of International Application No. PCT / US2015 / 02740 are incorporated herein by reference.

[0104] The mRNA vaccines described herein have been found to be superior to current vaccines in several ways. First, lipid nanoparticle (LNP) delivery is superior to other formulations described in the literature, including protamine-based vehicles, and does not require additional adjuvants. The use of LNPs allows for the effective delivery of chemically modified or unmodified mRNA vaccines. Furthermore, LNP-formulated mRNA vaccines, both modified and unmodified, have been shown herein to be significantly superior to conventional vaccines. In some embodiments, the mRNA vaccines of the present invention are at least 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1,000-fold superior to conventional vaccines.

[0105] Although attempts have been made to create functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic efficacy of these RNA vaccines has not yet been fully established. Surprisingly, in accordance with an embodiment of the present invention, the inventors have discovered a class of formulations for in vivo delivery of mRNA vaccines that result in significantly enhanced and in many ways synergistic immune responses, including increased antigen production and functional antibody production with neutralizing capacity. These results can be achieved even when extremely low doses of mRNA are administered compared to the doses of mRNA used in other types of lipid-based formulations. The formulations of the present invention have demonstrated unexpectedly significant in vivo immune responses sufficient to establish the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents. Furthermore, self-replicating RNA vaccines rely on viral replication pathways to deliver sufficient RNA to cells to produce an immunogenic response. The formulations of the present invention do not require viral replication to produce sufficient protein to produce a strong immune response. Therefore, the mRNAs of the present invention are not self-replicating RNA and do not contain components necessary for viral replication.

[0106] The present invention, in some aspects, relates to the surprising discovery that lipid nanoparticle (LNP) formulations significantly enhance the efficacy of mRNA vaccines, including chemically modified and unmodified mRNA vaccines. The efficacy of LNP-formulated mRNA vaccines was tested in vivo using several different antigens. The results presented herein demonstrate the unexpectedly superior efficacy of LNP-formulated mRNA vaccines over other commercially available vaccines.

[0107] In addition to producing an enhanced immune response, the formulations of the present invention produce a more rapid immune response at a lower dose than other vaccines tested. The mRNA-LNP formulations of the present invention also produce quantitatively and qualitatively better immune responses than vaccines formulated with different carriers.

[0108] The data described herein demonstrate that the formulations of the present invention provide unexpected and significant improvements over existing VZV antigen vaccines, including significantly higher levels of IgG production with chemically modified and unmodified mRNA VZV vaccines formulated in LNPs compared to VARIVAX and ZOSTAVAX. The onset of IgG production was significantly earlier with the chemically modified LNP mRNA vaccine than with the unmodified or commercially available vaccines tested.

[0109] Furthermore, the mRNA-LNP formulations of the present invention are superior to other vaccines, even when the mRNA dose is lower than that of other vaccines. The data show that all gE variant LNP mRNA vaccines induced significantly stronger immune responses than ZOSTAVAX® after two 10 μg doses and two 2 μg doses. When serum was diluted more than 100-fold, antibody titers were higher in the serum of VZV gE LNP mRNA-vaccinated mice than in the serum of ZOSTAVAX®-vaccinated mice, suggesting that the VZV gE LNP mRNA vaccine induced significantly stronger immune responses in mice than ZOSTAVAX®.

[0110] The results in mice were consistent with the immunogenicity observed in non-human primates. Rhesus macaques were primed with either the chemically modified VZV LNP mRNA vaccine or ZOSTAVAX®. The mRNA vaccine demonstrated higher anti-gE titers than ZOSTAVAX®, and unlike the ZOSTAVAX® group, CD4 T cells producing IFNγ, IL-2, or TNFα were produced at reasonable frequencies. The data also showed that a single dose of the mRNA vaccine after Zostavax exposure was equivalent to two doses of the mRNA vaccine in inducing similar T cell responses.

[0111] Some of the LNPs used in the studies described herein have been previously used in various animal models and humans to deliver siRNA. Given the observations made regarding siRNA delivery in LNP formulations, the fact that LNPs are useful for vaccines is quite surprising. It has been observed that therapeutic delivery of LNP-formulated siRNA causes undesirable inflammatory responses associated with transient IgM responses, typically leading to reduced antigen production and a weakened immune response. In contrast to the findings observed with siRNA, the LNP-mRNA formulations of the present invention are shown herein to produce an increase in IgG levels sufficient for prophylaxis and therapy, rather than a transient IgM response.

[0112] Nucleic Acids / Polynucleotides The varicella-zoster virus (VZV) vaccines provided herein comprise at least one (or more) ribonucleic acid (RNA, e.g., mRNA) polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide. The term "nucleic acid" in its broadest sense includes any compound and / or substance comprising polymers of nucleotides. These polymers are referred to as polynucleotides.

[0113] In some embodiments, at least one RNA polynucleotide of the VZV vaccine is encoded by at least one nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:41.

[0114] In some embodiments, at least one RNA (e.g., mRNA) polynucleotide of the VZV vaccine is encoded by at least one fragment (e.g., a fragment having an antigenic sequence or at least one epitope) of a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:41.

[0115] The nucleic acid (also referred to as polynucleotide) may be or may include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (including LNA, for example, LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functional group, and 2′-amino-α-LNA having a 2′-amino functional group), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or a chimera or combination thereof.

[0116] In some embodiments, polynucleotides of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (natural, non-natural, or modified amino acid polymer) that can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. Those of skill in the art will understand that, unless otherwise noted, the polynucleotide sequences described in this application use "T" when representing a DNA sequence, and that "T" is replaced with "U" when the sequence represents RNA (e.g., mRNA). Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number can include the corresponding RNA (e.g., mRNA) sequence encoded by that DNA, where each "T" in the DNA sequence is replaced with a "U."

[0117] The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a polyA tail. The polynucleotides of the present disclosure can function as mRNA, but their designed functional and / or structural features distinguish them from wild-type mRNA, and these features help to solve existing problems related to effective polypeptide expression using nucleic acid therapy.

[0118] In some embodiments, the RNA polynucleotide (e.g., mRNA) of the VZV vaccine encodes 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 antigenic polypeptides. In some embodiments, the RNA polynucleotide (e.g., mRNA) of the VZV RNA (e.g., mRNA) vaccine encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 antigenic polypeptides. In some embodiments, the RNA polynucleotide (e.g., mRNA) of the VZV vaccine encodes at least 100 antigenic polypeptides or at least 200 antigenic polypeptides. In some embodiments, the RNA polynucleotide (e.g., mRNA) of the VZV vaccine encodes 1 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 1 to 50, 1 to 100, 2 to 50, or 2 to 100 antigenic polypeptides.

[0119] In some embodiments, the polynucleotides (e.g., mRNA) of the present disclosure are codon-optimized. Codon optimization methods are known in the art and can be used as described herein. For example, any one or more of the sequences set forth in SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35, 39, 62, 66, 70, 74, 78, 82, 86, and 90, or any one or more of the sequences set forth in SEQ ID NOs: 92-108, can be codon-optimized. Codon optimization can be used, in some embodiments, to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to improve mRNA stability or reduce secondary structure, minimize tandemly repeated codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post-translational modification sites (e.g., glycosylation sites) to the encoded protein, add, remove, or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rates to allow proper folding of various domains of a protein, or reduce or eliminate problematic secondary structure within a polynucleotide. Codon optimization tools, algorithms, and services are known in the art; non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, an optimization algorithm is used to optimize the open reading frame (ORF) sequence.

[0120] In some embodiments, the codon-optimized sequence (e.g., the codon-optimized sequence of SEQ ID NOs: 92-108) shares less than 95% sequence identity to the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity to the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity to the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity to the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0121] In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity to the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding the polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, the codon-optimized sequence shares 65% to 75% or about 80% sequence identity to the native or wild-type sequence (e.g., the native or wild-type mRNA sequence encoding the polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0122] In some embodiments, the codon-optimized sequences (e.g., the codon-optimized sequences of SEQ ID NOs: 92-108) encode antigenic polypeptides that are as immunogenic as or more immunogenic (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more) than the antigenic polypeptides encoded by the (non-codon-optimized) sequences of SEQ ID NOs: 92-108.

[0123] In some embodiments, the VZV vaccine comprises an open reading frame encoding at least one VZV antigenic polypeptide with at least one modification, at least one RNA polynucleotide with at least one 5'-end cap, and is formulated into a lipid nanoparticle. 5'-capping of the polynucleotide can be achieved simultaneously during the in vitro transcription reaction by generating a 5'-guanosine cap structure using the following chemical RNA cap analogs according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of modified RNA can be achieved post-transcriptionally by using vaccinia virus capping enzyme to generate the "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure can be generated by using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure can be generated from the cap 1 structure by 2'-O-methylating the third 5'-nucleotide using 2'-O-methyltransferase. Cap 3 structure can be generated from the cap 2 structure by 2'-O-methylating the fourth 5'-nucleotide using 2'-O-methyltransferase. Enzymes may be derived from recombinant sources.

[0124] The modified mRNA has a stability of 12 to 18 hours or more than 18 hours, for example, 24, 36, 48, 60, 72 or more than 72 hours when transfected into mammalian cells.

[0125] In some embodiments, codon-optimized RNA can have an increased G / C level. The G / C content of a nucleic acid molecule (e.g., mRNA) can affect RNA stability. RNA with an increased amount of guanine (G) and / or cytosine (C) residues can be more functionally stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO 02 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modification in the coding region. Due to the degeneracy of the genetic code, this modification is made by replacing existing codons with codons that promote greater RNA stability without changing the resulting amino acid. This approach is limited to the coding region of the RNA.

[0126] Antigens / antigenic polypeptides In some embodiments, the antigenic polypeptide is a VZV glycoprotein. For example, the VZV glycoprotein can be VZV gE, gI, gB, gH, gK, gL, gC, gN, or gM, or an immunogenic fragment or epitope thereof. In some embodiments, the antigenic polypeptide is a VZV gE polypeptide. In some embodiments, the antigenic polypeptide is a VZV gI polypeptide. In some embodiments, the antigenic polypeptide is a VZV gB polypeptide. In some embodiments, the antigenic polypeptide is a VZV gH polypeptide. In some embodiments, the antigenic polypeptide is a VZV gK polypeptide. In some embodiments, the antigenic polypeptide is a VZV gL polypeptide. In some embodiments, the antigenic polypeptide is a VZV gC polypeptide. In some embodiments, the antigenic polypeptide is a VZV gN polypeptide. In some embodiments, the antigenic polypeptide is a VZV gM polypeptide.

[0127] In some embodiments, the antigenic polypeptide comprises two or more glycoproteins. The two or more glycoproteins may be encoded by a single RNA polynucleotide or by two or more RNA polynucleotides (e.g., each glycoprotein is encoded by a separate RNA polynucleotide). In some embodiments, the two or more glycoproteins may be any combination of VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of VZV gE and glycoproteins selected from gI, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of VZV gI and glycoproteins selected from VZV gE, gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins can be any combination of VZV gE, gI, and glycoproteins selected from gB, gH, gK, gL, gC, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more VZV glycoproteins are gE and gI. Alternative RNA vaccines comprising RNA polynucleotides encoding other viral protein components of VZV (e.g., tegument proteins) are also encompassed by the present disclosure. Accordingly, some embodiments of the present disclosure provide VZV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one VZV tegument protein, or an antigenic fragment or epitope thereof. In some embodiments, the antigenic polypeptide is a VZV tegument protein, or an antigenic fragment or epitope thereof.In other embodiments, the antigenic fragment(s) of the VZV vaccine may be at least one VZV tegument polypeptide and at least one VZV glycoprotein polypeptide, such as any VZV glycoprotein selected from gE, gI, gB, gH, gK, gL, gC, gN, and gM.

[0128] The present disclosure encompasses variant VZV antigenic polypeptides. In some embodiments, the variant VZV antigenic polypeptide is a variant VZV gE polypeptide. The variant VZV gE polypeptide is designed to avoid ER / Golgi apparatus retention of the polypeptide and result in increased surface expression of the antigen. In some embodiments, the variant gE polypeptide is truncated to remove the ER retention portion or cytoplasmic tail of the polypeptide. In some embodiments, the variant VZV gE polypeptide is mutated to reduce ER / Golgi apparatus / TGN localization of the VZV polypeptide. Such modifications inhibit ER trapping, thereby facilitating transport to the plasma membrane.

[0129] Thus, in some embodiments, the VZV glycoprotein is a variant gE polypeptide. VZV gE has a targeting sequence for the TGN at its C-terminus and is transported from the ER to the TGN in infected and gE-transfected cells. Most gE in the TGN appears to be retrieved by endocytosis from the plasma membrane, delivered to the TGN by endosomes, and then recycled to the plasma membrane. gE accumulates in the TGN along with other VZV proteins (e.g., tegument proteins) involved in the production of fully enveloped VZV virions. Therefore, mutations that reduce TGN localization and endocytosis promote the transport of gE to the plasma membrane.

[0130] A variant VZV gE polypeptide can be any truncated polypeptide lacking the anchor domain (ER retention domain). For example, a variant VZV gE polypeptide can be a truncated VZV gE polypeptide comprising at least amino acids 1-124 (e.g., including amino acids 1-124, 1-140, 1-160, 1-200, 1-250, 1-300, 1-350, 1-360, 1-400, 1-450, 1-500, 1-511, 1-550, and 1-561) and polypeptide fragments having fragment sizes within the recited size ranges. In one embodiment, a truncated VZV gE polypeptide comprises amino acids 1-561 of SEQ ID NO: 10. In some embodiments, a variant VZV gE polypeptide is a truncated polypeptide lacking the carboxy-terminal tail domain. Thus, in some embodiments, a truncated VZV gE polypeptide comprises amino acids 1-573 of SEQ ID NO: 10.

[0131] In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with ER retention, where the mutation(s) in the one or more motifs reduce retention of the VZV gE polypeptide in the ER and / or Golgi apparatus. In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more phosphorylated acidic motifs. For example, the variant VZV gE polypeptide can be a full-length VZV gE polypeptide having a Y582G mutation, a Y569A mutation, or both a Y582G mutation and a Y569A mutation. Alternatively, the variant VZV gE polypeptide can be an antigenic fragment, for example, comprising amino acids 1-573 of VZV gE and having a Y569A mutation. Alternatively, the variant VZV gE polypeptide can be an antigenic fragment having a mutation in an acidic phosphorylated motif, such as an SST motif. For example, the variant VZV gE polypeptide can be an antigenic fragment having the sequence AEAADA (SEQ ID NO: 58).

[0132] In some embodiments, the variant VZV gE polypeptide is a full-length VZV gE polypeptide having an additional sequence at the C-terminus that promotes secretion of the polypeptide. For example, the variant VZV gE polypeptide can be a full-length VZV gE polypeptide having an Igκ sequence at the C-terminus. In some embodiments, the VZV gE polypeptide has an additional sequence at the C-terminus that promotes secretion (having an Igκ sequence at the C-terminus), and the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with ER retention, TGN localization, and / or endocytosis (e.g., a Y582G mutation, a Y569A mutation, or both the Y582G and Y569A mutations), and / or has at least one mutation in one or more phosphorylated acidic motifs. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide that lacks the anchor domain (ER retention domain) and has an additional sequence at the C-terminus that promotes secretion of the polypeptide (e.g., having an Igκ sequence at the C-terminus). In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1-561 of SEQ ID NO: 10 and has an Igκ sequence at the C-terminus. In some embodiments, the variant VZV gE polypeptide is a truncated polypeptide that lacks a carboxy-terminal tail domain and has an additional sequence at the C-terminus that promotes secretion of the polypeptide (e.g., has an Igκ sequence at the C-terminus). In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1-573 of SEQ ID NO: 10 and has an Igκ sequence at the C-terminus.

[0133] In some embodiments, a VZV antigenic polypeptide is longer than 25 amino acids and shorter than 50 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides may be single molecules or multimolecular complexes, such as dimers, trimers, or tetramers. Polypeptides may also include single-chain polypeptides or multi-chain polypeptides, such as antibodies or insulin, which may be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.

[0134] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least 50% identity to the native or reference sequence. In some embodiments, variants share at least 80% or at least 90% identity with the native or reference sequence.

[0135] In some embodiments, a "variant mimetic" is provided. As used herein, a "variant mimetic" contains at least one amino acid that can mimic an activating sequence. For example, glutamate can act as a mimic of phosphorylated threonine and / or phosphorylated serine. Alternatively, a variant mimetic can result in inactivation or an inactivated product containing the mimetic. For example, phenylalanine can act as an inactivating substitute for tyrosine, and alanine can act as an inactivating substitute for serine.

[0136] "Orthologs" refer to genes in different species that evolved from a common ancestral gene through speciation. Orthologs typically retain the same function during evolution. Identifying orthologs is important for reliable prediction of gene function in newly sequenced genomes.

[0137] "Analog" is meant to include polypeptide variants that differ by one or more amino acid alterations, e.g., substitution, addition, or deletion of an amino acid residue, but which still retain one or more properties of the parent or starting polypeptide.

[0138] "Paralogues" are genes (or proteins) related by duplication within a genome. Orthologs retain the same function during evolution, while paralogs have evolved new, albeit related, functions.

[0139] The present disclosure provides several types of polynucleotide- or polypeptide-based compositions, including variants and derivatives. These include, for example, substitutional, insertional, deletional, and covalent variants and derivatives. The term "derivative" is used interchangeably with the term "variant," but generally refers to a molecule that has been modified and / or changed in any way compared to a reference or starting molecule.

[0140] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly a polypeptide sequence disclosed herein, are within the scope of this disclosure. For example, a sequence tag or one or more amino acids, such as lysine, can be added to the peptide sequence (e.g., at the N- or C-terminus). The sequence tag can be used for peptide detection, purification, or location. Lysine can be used to increase the solubility of the peptide or to enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the peptide or protein amino acid sequence can be optionally deleted to provide a truncated sequence. Alternatively, specific amino acids (e.g., C- or N-terminal residues) can be deleted depending on the intended use of the sequence, e.g., to express the sequence as part of a larger sequence that is soluble or linked to a solid support. In alternative embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., foldon regions), and the like, can be replaced with alternative sequences that achieve the same or similar functions. Such sequences are readily identifiable by one of ordinary skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N- or C-terminus) that can be deleted, for example, immediately prior to preparation of an RNA (e.g., mRNA) vaccine.

[0141] "Substitutional variants" with respect to polypeptides are those in which at least one amino acid residue in a native or starting sequence has been deleted and a different amino acid inserted in its place in the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0142] As used herein, the term "conservative amino acid substitution" refers to a substitution in which an amino acid normally present in a sequence is replaced with another amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of nonpolar (hydrophobic) residues such as isoleucine, valine, and leucine for another nonpolar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another polar residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, the substitution of a basic residue such as lysine, arginine, or histidine for another basic residue, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue, are further examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a nonpolar residue.

[0143] A "feature" with respect to a polypeptide or polynucleotide is defined as a distinct amino acid sequence-based or nucleotide-based component of the molecule, respectively. Features of a polypeptide encoded by a polynucleotide include surface expression, local conformational shape, fold, loop, half-loop, domain, half-domain, site, terminus, or any combination thereof.

[0144] The term "domain," as used herein with respect to polypeptides, refers to a polypeptide motif having one or more identifiable structural or functional features or characteristics (e.g., binding ability, functioning as a site of protein-protein interaction).

[0145] As used herein with respect to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when it relates to amino acid-based embodiments. As used herein with respect to polypeptides, the term "site" is used synonymously with "nucleotide" when it relates to nucleotide-based embodiments. A site represents a position within a peptide or polypeptide or polynucleotide that can be modified, manipulated, altered, derivatized, or changed within a polypeptide- or polynucleotide-based molecule.

[0146] As used herein with respect to a polypeptide or polynucleotide, the term "terminus" refers to the end of the polypeptide or polynucleotide, respectively. Such terminus is not limited to the initial or final position of the polypeptide or polynucleotide, but may include amino acids or nucleotides added to the terminal region. Polypeptide-based molecules can be characterized as having both an N-terminus (ending with an amino acid having a free amino group (NH)) and a C-terminus (ending with an amino acid having a free carboxyl group (COOH)). Proteins are sometimes composed of multiple polypeptide chains (multimers, oligomers) held together by disulfide bonds or noncovalent forces. These proteins have multiple N- and C-termini. Alternatively, the termini of polypeptides may be modified to begin or end with non-polypeptide-based moieties, such as organic conjugates, as appropriate.

[0147] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptide of interest. For example, any protein fragment of a reference protein (i.e., a polypeptide sequence at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical) that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length is provided herein. In another example, any protein containing a stretch of 20, 30, 40, 50, or 100 amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the present disclosure. In some embodiments, the polypeptide contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as indicated, of any of the sequences described or referenced herein. In some embodiments, the protein fragment is longer than 25 amino acids and shorter than 50 amino acids.

[0148] A polypeptide or polynucleotide molecule of the present disclosure may share a degree of sequence similarity or sequence identity with a reference molecule (e.g., a reference polypeptide or reference polynucleotide), such as a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). The term "identity," as known in the art, refers to the relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, identity also refers to the degree of sequence relatedness between sequences, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences with gap alignment (if present), as processed by a particular mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be readily calculated by known methods. "Percent identity," as applied to a polypeptide or polynucleotide sequence, is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. Identity is dependent on the calculation of percent identity, with the understanding that values ​​may vary depending on gaps and penalties introduced into the calculation. Generally, a particular polynucleotide or polypeptide variant will have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to a particular reference polynucleotide or polypeptide sequence, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.Such alignment tools include those in the BLAST suite (Stephen F. Altschul, et al., (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another widely used local alignment method is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common molecular subsequences", J. Mol. Biol. 147:195-197). A common global alignment method based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol. Biol. 48:443-453). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0149] As used herein, the term "homology" refers to the overall relatedness between polymers, e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymers (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity as determined by alignment of matching residues are called homologous. Homology is a qualitative term that describes the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymers 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% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical over at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by their ability to encode a uniquely specified stretch of at least 4-5 amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a uniquely specified stretch of at least 4-5 amino acids. Two protein sequences are considered to be homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical over at least one stretch of at least 20 amino acids.

[0150] Homology suggests that the compared sequences evolved from a common evolutionary origin. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) that is related to a second amino acid or nucleic acid sequence by being derived from a common ancestral sequence. The term "homolog" can apply to the relationship between genes and / or proteins that were separated by a speciation event or by a gene duplication event.

[0151] Multiprotein and multicomponent vaccines The present disclosure encompasses VZV vaccines comprising multiple RNA (e.g., mRNA) polynucleotides, each encoding a single antigenic polypeptide, as well as VZV vaccines comprising a single RNA polynucleotide encoding more than one antigenic polypeptide (e.g., as a fusion polypeptide). Thus, a vaccine composition comprising an RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a first VZV antigenic polypeptide and an RNA polynucleotide (e.g., mRNA) having an open reading frame encoding a second VZV antigenic polypeptide will be understood to encompass (a) vaccines comprising a first RNA polynucleotide encoding the first VZV antigenic polypeptide and a second RNA polynucleotide encoding the second VZV antigenic polypeptide, and (b) vaccines comprising a single RNA polynucleotide encoding the first and second VZV antigenic polypeptides (e.g., as a fusion polypeptide). The VZV RNA vaccines of the present disclosure, in some embodiments, comprise two to ten (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more RNA polynucleotides, each having an open reading frame encoding a different VZV antigenic polypeptide (or a single RNA polynucleotide encoding two to ten or more different VZV antigenic polypeptides).In some embodiments, the VZV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding a VZV gE protein, an RNA polynucleotide having an open reading frame encoding a VZV gI protein, an RNA polynucleotide having an open reading frame encoding a VZV gB protein, an RNA polynucleotide having an open reading frame encoding a VZV gH protein, an RNA polynucleotide having an open reading frame encoding a VZV gK protein, an RNA polynucleotide having an open reading frame encoding a VZV gL protein, an RNA polynucleotide having an open reading frame encoding a VZV gC protein, an RNA polynucleotide having an open reading frame encoding a VZV gN protein, and an RNA polynucleotide having an open reading frame encoding a VZV gM protein. In some embodiments, the VZV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding a VZV gE protein and an RNA polynucleotide having an open reading frame encoding a VZV gI protein. In some embodiments, the VZV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding a VZV gE protein or a gE variant.

[0152] In some embodiments, the RNA polynucleotide encodes a VZV antigenic polypeptide fused to a signal peptide (e.g., SEQ ID NO: 56, 57, 109, 110, or 111). The signal peptide can be fused to the N-terminus or C-terminus of the antigenic polypeptide.

[0153] signal peptide In some embodiments, the antigenic polypeptide encoded by a VZV polynucleotide comprises a signal peptide. Signal peptides comprise an N-terminal 15-60 amino acid protein, typically required for translocation across membranes in the secretory pathway, and thus universally control the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. Signal peptides generally consist of three regions: an N-terminal region of variable length (usually containing positively charged amino acids), a hydrophobic region, and a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs ribosomes to the rough endoplasmic reticulum (ER) membrane and initiates transport of the elongating peptide chain across the membrane. However, the signal peptide is not responsible for the final destination of the mature protein. Secretory proteins lacking additional addressing tags in their sequence are normally secreted into the external environment. The signal peptide is either cleaved from the precursor protein by signal peptidases present in the endoplasmic reticulum (ER) or remains intact and functions as a membrane anchor. In recent years, a more refined view of signal peptides has emerged, showing that the function and immunodominance of certain signal peptides is much more diverse than previously anticipated.

[0154] Signal peptides typically function to facilitate targeting of newly synthesized proteins to the endoplasmic reticulum (ER) for processing. ER processing produces mature envelope proteins, where the signal peptide is typically cleaved by a host cell signal peptidase. Signal peptides can also facilitate targeting of proteins to the cell membrane. The VZV vaccines of the present disclosure can include, for example, an RNA polynucleotide encoding an artificial signal peptide, where the sequence encoding the signal peptide is operably linked to and in-frame with the coding sequence of a VZV antigenic polypeptide. Thus, in some embodiments, the VZV vaccines of the present disclosure produce antigenic polypeptides comprising a VZV antigenic polypeptide fused to a signal peptide. In some embodiments, the signal peptide is fused to the N-terminus of the VZV antigenic polypeptide. In some embodiments, the signal peptide is fused to the C-terminus of the VZV antigenic polypeptide.

[0155] In some embodiments, the signal peptide fused to the VZV antigenic polypeptide is an artificial signal peptide. In some embodiments, the artificial signal peptide fused to the VZV antigenic polypeptide encoded by a VZV RNA (e.g., mRNA) vaccine is derived from an immunoglobulin protein, such as an IgE signal peptide or an IgG signal peptide. In some embodiments, the signal peptide fused to the VZV antigenic polypeptide encoded by a VZV RNA (e.g., mRNA) vaccine is an Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 56). In some embodiments, the signal peptide fused to the VZV antigenic polypeptide encoded by a VZV RNA (e.g., mRNA) vaccine is an IgGk chain V-III region HAH signal peptide (IgGkSP) having the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 57). In some embodiments, the VZV antigenic polypeptide encoded by the VZV RNA (e.g., mRNA) vaccine has an amino acid sequence set forth in one of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, and 45-55 fused to a signal peptide of any of SEQ ID NOs: 56, 57, 109, 110, and 111. The examples disclosed herein are not intended to be limiting, and any signal peptide known in the art to facilitate targeting of proteins to the ER and / or targeting of proteins to the plasma membrane for processing can be used in accordance with the present disclosure.

[0156] The signal peptide may be 15 to 60 amino acids in length, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length. In some embodiments, the signal peptide is 20 to 60, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to 60, 55 to 60, 15 to 55, 20 to 55, 25 to 55, 30 to 55, 35 to 55, 40 to 55, 45 to 55, 50 to 55, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, It may have a length of 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25 or 15-20 amino acids.

[0157] The signal peptide is typically cleaved from the nascent polypeptide at a cleavage site during ER processing. The mature VZV antigenic polypeptides produced by the VZV RNA vaccines of the present disclosure typically do not include the signal peptide.

[0158] chemical modification The RNA (e.g., mRNA) vaccines of the present disclosure, in some embodiments, comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one respiratory syncytial virus (VZV) antigenic polypeptide, wherein the RNA comprises at least one chemical modification.

[0159] The terms "chemically modified" and "chemically modified" refer to modifications to adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides in at least one of their position, pattern, percentage, or population. Generally, these terms do not refer to naturally occurring ribonucleotide modifications in the 5'-end mRNA cap portion.

[0160] Modifications of polynucleotides include, but are not limited to, those described herein, including chemical modifications. Polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides) can contain modifications that are natural, non-natural, or polynucleotides can contain a combination of natural and non-natural modifications. Polynucleotides can contain any useful modification, for example, modifications of the sugar, nucleobase, or internucleoside linkage (e.g., phosphate, phosphodiester linkage, or linkage to a phosphodiester backbone).

[0161] With respect to polypeptides, the term "modified" refers to modifications to the standard set of 20 amino acids. The polypeptides provided herein are also considered "modified" if they contain amino acid substitutions, insertions, or a combination of substitutions and insertions.

[0162] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises a variety (more than one) different modifications. In some embodiments, specific regions of a polynucleotide contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), when introduced into a cell or organism, exhibits reduced degradation in the cell or organism, respectively, compared to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), when introduced into a cell or organism, may exhibit reduced immunogenicity (e.g., a reduced innate response) in the cell or organism, respectively.

[0163] Polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides) in some embodiments include non-naturally occurring modified nucleotides introduced during or after the synthesis of the polynucleotide to achieve a desired function or property. Modifications may occur in the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced at the ends of the strand or elsewhere in the strand using chemical synthesis or polymerase enzymes. Any region of a polynucleotide can be chemically modified.

[0164] The present disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides). A "nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly, to contain one or more modified or non-natural nucleosides. A polynucleotide can contain one or more regions of linked nucleosides. Such regions can have various backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide would contain a region of nucleotides.

[0165] Base pairing of modified nucleotides encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard base and a standard base, or between two complementary non-standard base structures (e.g., in polynucleotides having at least one chemical modification). One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of the present disclosure.

[0166] Modifications of polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides) useful in the compositions, vaccines, methods, and synthetic procedures of the present disclosure, including but not limited to chemical modifications, include, but are not limited to, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, 1,2'-O-dimethyladenosine, 1-methyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), 2 ... , 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, N6,N6-dimethyladenosine, N6-acetyladenosine, N6-hydroxynorvalylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, 2-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 7-deazaadenosine, N1-methyl-adenosine, N6,N6(dimethyl)adenine, N6-cis-hydroxy-isopentenyl-adenosine, α-thio-adenosine, 2(amino)adenine, 2(aminopropyl)adenine, 2(methylthio)N6(isopentenyl)adenine, 2-(alkyl)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(halo)adenine, 2-(halo)adenine, 2-(propyl)adenine, 2'-amino-2'-deoxy-ATP, 2' -azido-2'-deoxy-ATP, 2'-deoxy-2'-α-aminoadenosine TP, 2'-deoxy-2'-α-azidoadenosine TP, 6(alkyl)adenine, 6(methyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7(deaza)adenine, 8(alkenyl)adenine, 8(alkynyl)adenine, 8(amino)adenine, 8(thioalkyl)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, 8-azido-adenosine, azaadenine, deazaadenine, N6(methyl)adenine, N6-(isopentyl)adenine, 7-deaza-8-aza-adenosine, 7-methyladenine, 1-deazaadenosine TP, 2'fluoro-N6-Bz-deoxyadenosine TP, 2' -OMe-2-amino-ATP, 2'O-methyl-N6-Bz-deoxyadenosine TP, 2'-α-ethynyl adenosine TP, 2-aminoadenine, 2-amino adenosine TP, 2-amino-ATP, 2'-α-trifluoromethyl adenosine TP, 2-azidoadenosine TP, 2'-β-ethynyl adenosine TP, 2-bromoadenosine TP, 2'-β-trifluoromethyl adenosine TP, 2-chloroadenosine TP, 2'-deoxy-2',2'-Difluoroadenosine TP, 2'-deoxy-2'-α-mercaptoadenosine TP, 2'-deoxy-2'-α-thiomethoxyadenosine TP, 2'-deoxy-2'-β-aminoadenosine TP, 2'-deoxy-2'-β-azidoadenosine TP, 2'-deoxy-2'-β-bromoadenosine TP, 2'-deoxy-2'-β-chloroadenosine TP, 2'-deoxy-2'-β-fluoroadenosine TP, 2'-deoxy-2'-β-iodoadenosine TP , 2'-deoxy-2'-b-mercaptoadenosine TP, 2'-deoxy-2'-b-thiomethoxyadenosine TP, 2-fluoroadenosine TP, 2-iodoadenosine TP, 2-mercaptoadenosine TP, 2-methoxy-adenine, 2-methylthio-adenine, 2-trifluoromethyladenosine TP, 3-deaza-3-bromoadenosine TP, 3-deaza-3-chloroadenosine TP, 3-deaza-3-fluoroadenosine TP, 3-deaza-3-iodoadenosine TP, 3-deazaadenosine TP, 4'-azidoadenosine TP, 4'-carbocyclic adenosine TP, 4'-ethynyl adenosine TP, 5'-homo-adenosine TP, 8-aza-ATP, 8-bromo-adenosine TP, 8-trifluoromethyl adenosine TP, 9-deazaadenosine TP, 2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 7-deaza-8-aza-2-aminopurine, 2,6-diaminopurine, 7-deaza Aza-8-aza-adenine, 7-deaza-2-aminopurine, 2-thiocytidine, 3-methylcytidine, 5-formylcytidine, 5-hydroxymethylcytidine, 5-methylcytidine, N4-acetylcytidine, 2'-O-methylcytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, lysidine, N4,2'-O-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N4-methylcytidine, N4,N4-dimethyl-2'-OMe-cytidine TP, 4-methylcytidine, 5-aza-cytidine, pseudo-iso-cytidine, pyrrolo-cytidine, α-thio-cytidine, 2-(thio)cytosine, 2'-amino-2'-deoxy-CTP, 2'-azido-2'-deoxy-CTP, 2'-deoxy-2'-α-aminocytidine TP, 2'-deoxy-2'-α-azidocytidine TP, 3(deaza)5(aza)cytosine, 3(methyl)cytosine, 3-(alkyl)cytosine, 3-(deaza)5(aza)cytosine, -(Methyl)cytidine, 4,2'-O-dimethylcytidine, 5(halo)cytosine, 5(methyl)cytosine, 5(propynyl)cytosine, 5(trifluoromethyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-bromo-cytidine, 5-iodo-cytidine, 5-propynylcytosine, 6-(azo)cytosine, 6-aza-cytidine, azacytosine, deazacytosine, N4(acetyl)cytosine Syn, 1-methyl-1-deaza-pseudoisocytidine, 1-methyl-pseudoisocytidine, 2-methoxy-5-methyl-cytidine, 2-methoxy-cytidine, 2-thio-5-methyl-cytidine, 4-methoxy-1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-pseudoisocytidine, 5-aza-zebularine, 5-methyl-zebularine, pyrrolo-pseudoisocytidine Doisocytidine, Zebularine, (E)-5-(2-bromo-vinyl)cytidine TP, 2,2'-anhydro-cytidine TP hydrochloride, 2'fluoro-N4-Bz-cytidine TP, 2'fluoro-N4-acetyl-cytidine TP, 2'-O-methyl-N4-acetyl-cytidine TP, 2'O-methyl-N4-Bz-cytidine TP, 2'-a-ethynylcytidine TP, 2'-a-trifluoromethylcytidine TP, 2'-b-ethynylcytidine TP, 2'-b-trifluoromethylcytidine TP, 2'-deoxy-2',2'-Difluorocytidine TP, 2'-deoxy-2'-α-mercaptocytidine TP, 2'-deoxy-2'-α-thiomethoxycytidine TP, 2'-deoxy-2'-β-aminocytidine TP, 2'-deoxy-2'-β-azidocytidine TP, 2'-deoxy-2'-β-bromocytidine TP, 2'-deoxy-2'-β-chlorocytidine TP, 2'-deoxy-2'-β-fluorocytidine TP, 2'-deoxy-2'-β-iodocytidine TP, 2 '-Deoxy-2'-b-mercaptocytidine TP, 2'-deoxy-2'-b-thiomethoxycytidine TP, 2'-O-methyl-5-(1-propynyl)cytidine TP, 3'-ethynylcytidine TP, 4'-azidocytidine TP, 4'-carbocyclic cytidine TP, 4'-ethynylcytidine TP, 5-(1-propynyl)ara-cytidine TP, 5-(2-chloro-phenyl)-2-thiocytidine TP, 5-(4-amino-phenyl)-2-thiocytidine TP, 5 -aminoallyl-CTP, 5-cyanocytidine TP, 5-ethynylalanine-cytidine TP, 5-ethynylcytidine TP, 5'-homocytidine TP, 5-methoxycytidine TP, 5-trifluoromethyl-cytidine TP, N4-aminocytidine TP, N4-benzoylcytidine TP, pseudoisocytidine, 7-methylguanosine, N2,2'-O-dimethylguanosine, N2-methylguanosine, wyosine, 1,2'-O-dimethylguanosine, 1-methylguanosine, Chiruguanosine, 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, archaeosine, methylwiosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, 6-thio-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, N1-methyl-guanosine, α-thio-guanosine, 2(propyl)guanine, 2-(alkyl)guanine, 2'-amino-2'-deoxy-GTP, 2'-azido-2'-deoxy-GTP, 2'-deoxy-2'-α-aminoguanosine TP, 2'-deoxy-2'-α-azidoguanosine TP, 6(methyl)guanosine, 6-(azido)guanosine (alkyl)guanine, 6-(methyl)guanine, 6-methyl-guanosine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(halo)guanine, 8-(thioalkyl)guanine, 8-(alkenyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, azaguanine, deazaguanine, N(methyl)guanine, N-(methyl)guanine, 1-methyl-6-thio-guanosine, 6-methoxy-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7-methyl-guanosine, 8-oxo-guanosine, N2,N2-dimethyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, 1-Me-GTP, 2'fluoro-N2-isobutyl-guanosine TP, 2'O-methyl-N2-isobutyl-guanosine TP, 2'-a-ethynylguanosine TP, 2'-a-trifluoromethylguanosine TP, 2'-b-ethynylguanosine TP, 2'-b-trifluoromethylguanosine TP, 2'-deoxy-2',2'-Difluoroguanosine TP, 2'-deoxy-2'-α-mercaptoguanosine TP, 2'-deoxy-2'-α-thiomethoxyguanosine TP, 2'-deoxy-2'-β-aminoguanosine TP, 2'-deoxy-2'-β-azidoguanosine TP, 2'-deoxy-2'-β-bromoguanosine TP, 2'-deoxy-2'-β-chloroguanosine TP, 2'-deoxy-2'-β-fluoroguanosine TP, 2'-deoxy-2'-β-iodoguanosine TP, 2'-deoxy-2'-β-mercaptoguanosine TP, 2'-deoxy-2'-, b-Thiomethoxyguanosine TP, 4'-azidoguanosine TP, 4'-carbocyclic guanosine TP, 4'-ethynylguanosine TP, 5'-homoguanosine TP, 8-bromoguanosine TP, 9-deazaguanosine TP, N2-isobutylguanosine TP, 1-methylinosine, inosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 7-methylinosine, 2'-O-methylinosine, epoxyqueosine, galactosylqueosine, mannosilqueosine, queosine, allylamino-thymidine, azathymidine, deazathymidine, deoxythymidine, 2'-O-methyluridine, 2-thiouridine, 3-methyluridine, 5-carboxymethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-tauricin 2'-O-methyluridine, 5-taurinomethyluridine, dihydrouridine, pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, 1-methylpseudouridine, 1-ethyl-pseudouridine, 2'-O-methyluridine, 2'-O-methylpseudouridine, 2'-O-methyluridine, 2-thio-2'-O-methyluridine, 3-(3-amino-3-carboxypropyl)uridine, 3,2'-O-dimethyluridine, 3-methyl-pseudouridine TP, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine, 5,6-Dihydro-uridine, 5-aminomethyl-2-thiouridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carbamoylmethyluridine Ilmethyluridine TP, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methyluridine, 5-methoxyuridine, 5-methyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methyldihydrouridine, 5-oxyacetic acid-uridine TP, 5-oxyacetic acid-methyl ester-uridine TP, N1-methyl-pseudo-uracil , N1-ethyl-pseudo-uracil, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 3-(3-amino-3-carboxypropyl)-uridine TP, 5-(iso-pentenylaminomethyl)-2-thiouridine TP, 5-(iso-pentenylaminomethyl)-2'-O-methyluridine TP, 5-(iso-pentenylaminomethyl)uridine TP, 5-propynyluracil, α-thio-uridine, 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-2(thio)-uridine ... 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-4(thio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-substituted 4(thio)pseudouracil, 1-substituted pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP, 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP, 1-methyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 2(thio)pseudouracil, 2'-deoxyuridin uridine, 2'-fluorouridine, 2-(thio)uracil, 2,4-(dithio)pseudouracil, 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-guanosine, 2'-amino-2'-deoxy-UTP, 2'-azido-2'-deoxy-UTP, 2'-azido-deoxyuridine TP, 2'-O-methylpseudouridine, 2'-deoxyuridine, 2'-fluorouridine, 2'-deoxy-2'-α-aminouridine TP, 2'-deoxy-2'-α-azidouridine TP, 2-methyl Chilpseudouridine, 3(3-amino-3-carboxypropyl)uracil, 4(thio)pseudouracil, 4-(thio)pseudouracil, 4-(thio)uracil, 4-thiouracil, 5(1,3-diazol-1-alkyl)uracil, 5(2-aminopropyl)uracil, 5(aminoalkyl)uracil, 5(dimethylaminoalkyl)uracil, 5(guanidinium alkyl)uracil, 5(methoxycarbonylmethyl)-2-(thio)uracil, 5(methoxycarbonyl -methyl)uracil, 5(methyl)2(thio)uracil, 5(methyl)2,4(dithio)uracil, 5(methyl)4(thio)uracil, 5(methylaminomethyl)-2(thio)uracil, 5(methylaminomethyl)-2,4(dithio)uracil, 5(methylaminomethyl)-4(thio)uracil, 5(propynyl)uracil, 5(trifluoromethyl)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(alkyl)-2,4(dithio)pseudouracil, 5-(alkyl)-4(thio)pseudouracil, 5-(alkyl)pseudouracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(halo)uracil, 5-(l,3-diazol-l-alkyl)uracil, 5-(methoxy)uracil, 5-( 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(methyl)2(thio)uracil, 5-(methyl)2,4(dithio)uracil, 5-(methyl)4(thio)uracil, 5-(methyl)-2-(thio)pseudouracil, 5-(methyl)-2,4(dithio)pseudouracil, 5-(methyl)-4(thio)pseudouracil, 5-(methyl)pseudouracil, 5-(methylaminomethyl)-2(thio)uracil, 5-(methylaminomethyl)-2,4(dithio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 5-aminoallyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-uracil, 6(azo)uracil, 6-(azo)uracil, 6-aza-uridine, allylamino-uracil, azauracil, deazauracil, N3(methyl)uracil, pseudo-UTP-1-2-ethanoic acid, pseudouracil, 4-thio-pseudo-UTP, 1-carbo 2-Methoxymethyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 1-propynyl-uridine, 1-taurinomethyl-1-methyl-uridine, 1-taurinomethyl-4-thio-uridine, 1-taurinomethyl-pseudouridine, 2-methoxy-4-thio-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio -pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, (±)1-(2-hydroxypropyl)pseudouridine TP, (2R)-1-(2-hydroxypropyl)pseudouridine TP, (2S)-1-(2-hydroxypropyl)pseudouridine TP, (E)-5-(2-bromo-vinyl)ara-uridine TP, (E)-5 -(2-Bromo-vinyl)uridine TP, (Z)-5-(2-Bromo-vinyl)ara-uridine TP, (Z)-5-(2-Bromo-vinyl)uridine TP, 1-(2,2,2-trifluoroethyl)-pseudo-UTP, 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP, 1-(2,2-diethoxyethyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudo-UTP, 1-(2,4,6-trimethyl-phenyl)pseudo-UTP, 1-(2-amino-2-carboxyethyl)pseudo-UTP, 1-(2-amino-ethyl)pseudo-UTP, 1-(2-hydroxyethyl)pseudouridine TP, 1-(2-ethoxyethyl)pseudouridine TP, 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine TP, 1-(3,4-dimethoxybenzyl)pseudouridine TP, 1-(3-amino-3-carboxypropyl)pseudouridine TP pseudo-UTP, 1-(3-amino-propyl)pseudo-UTP, 1-(3-cyclopropyl-prop-2-ynyl)pseudouridine TP, 1-(4-amino-4-carboxybutyl)pseudo-UTP, 1-(4-amino-benzyl)pseudo-UTP, 1-(4-amino-butyl)pseudo-UTP, 1-(4-amino-phenyl)pseudo-UTP, 1-(4-azidobenzyl)pseudouridine TP, 1-(4-bromobenzyl)pseudouridine TP, 1-(4 -chlorobenzyl)pseudouridine TP, 1-(4-fluorobenzyl)pseudouridine TP, 1-(4-iodobenzyl)pseudouridine TP, 1-(4-methanesulfonylbenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudo-UTP, 1-(4-methoxyphenyl)pseudo-UTP, 1-(4-methylbenzyl)pseudouridine TP, 1-(4-methylbenzyl)pseudouridine TP pseudo-UTP, 1-(4-nitrobenzyl)pseudo-UTP, 1-(4-nitro-benzyl)pseudo-UTP, 1(4-nitro-phenyl)pseudo-UTP, 1-(4-thiomethoxybenzyl)pseudouridine TP, 1-(4-trifluoromethoxybenzyl)pseudouridine TP, 1-(4-trifluoromethylbenzyl)pseudouridine TP, 1-(5-amino-pentyl)pseudo-UTP, 1-(6-amino-hexyl)pseudo-UTP, 1,6-Dimethyl-pseudo-UTP, 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP, 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine TP, 1-acetylpseudouridine TP, 1-alkyl-6-(1-propynyl)-pseudo-UTP, 1-alkyl-6-(2-propynyl)-pseudo-UT P, 1-alkyl-6-allyl-pseudo-UTP, 1-alkyl-6-ethynyl-pseudo-UTP, 1-alkyl-6-homoallyl-pseudo-UTP, 1-alkyl-6-vinyl-pseudo-UTP, 1-allylpseudouridine TP, 1-aminomethyl-pseudo-UTP, 1-benzoylpseudouridine TP, 1-benzyloxymethylpseudouridine TP, 1-benzyl-pseudo-UTP, 1-biotinyl-, PEG2-pseudouridine TP, 1-biotinylpseudouridine TP, 1-butyl-pseudo-UTP, 1-cyanomethylpseudouridine TP, 1-cyclobutylmethyl-pseudo-UTP, 1-cyclobutyl-pseudo-UTP, 1-cycloheptylmethyl-pseudo-UTP, 1-cycloheptyl-pseudo-UTP, 1-cyclohexylmethyl-pseudo-UTP, 1-cyclohexyl-pseudo-UTP, 1-cyclooctylmethyl-pseudo-UTP, 1-cyclooctyl-pseudo-UTP, 1-cyclopentylmethyl-pseudo-UTP pseudo-UTP, 1-cyclopentyl-pseudo-UTP, 1-cyclopropylmethyl-pseudo-UTP, 1-cyclopropyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 1-hexyl-pseudo-UTP, 1-homoallylpseudouridine TP, 1-hydroxymethylpseudouridine TP, 1-isopropyl-pseudo-UTP, 1-Me-2-thio-pseudo-UTP, 1-Me-4-thio-pseudo-UTP, 1-Me-alpha-thio-pseudo-UTP, 1-methanesulfonylmethylpseudouridine TP, 1-methoxy Methylpseudouridine TP, 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP, 1-methyl-6-(4-morpholino)-pseudo-UTP, 1-methyl-6-(4-thiomorpholino)-pseudo-UTP, 1-methyl-6-(substituted phenyl)pseudo-UTP, 1-methyl-6-amino-pseudo-UTP, 1-methyl-6-azido-pseudo-UTP, 1-methyl-6-bromo-pseudo-UTP, 1-methyl-6-butyl-pseudo-UTP, 1-methyl-6-chloro-pseudo-UTP, 1-methyl-6-cyano -pseudo-UTP, 1-methyl-6-dimethylamino-pseudo-UTP, 1-methyl-6-ethoxy-pseudo-UTP, 1-methyl-6-ethylcarboxylate-pseudo-UTP, 1-methyl-6-ethyl-pseudo-UTP, 1-methyl-6-fluoro-pseudo-UTP, 1-methyl-6-formyl-pseudo-UTP, 1-methyl-6-hydroxyamino-pseudo-UTP, 1-methyl-6-hydroxy-pseudo-UTP, 1-methyl-6-iodo-pseudo-UTP, 1-methyl-6-isopropyl-pseudo-UTP,1-methyl-6-methoxy-pseudo-UTP, 1-methyl-6-methylamino-pseudo-UTP, 1-methyl-6-phenyl-pseudo-UTP, 1-methyl-6-propyl-pseudo-UTP, 1-methyl-6-tert-butyl-pseudo-UTP, 1-methyl-6-trifluoromethoxy-pseudo-UTP, 1-methyl-6-trifluoromethyl-pseudo-UTP, 1-morpholinomethylpseudouridine TP, 1-pentyl-pseudo-UTP, 1-phenyl-pseudo-UTP, 1-pivaloylpseudouridine TP, 1-propargyl Lupseudouridine TP, 1-propyl-pseudo-UTP, 1-propynyl-pseudouridine, 1-p-tolyl-pseudo-UTP, 1-tert-butyl-pseudo-UTP, 1-thiomethoxymethylpseudouridine TP, 1-thiomorpholinomethylpseudouridine TP, 1-trifluoroacetylpseudouridine TP, 1-trifluoromethyl-pseudo-UTP, 1-vinylpseudouridine TP, 2,2'-anhydro-uridine TP, 2'-bromo-deoxyuridine TP, 2'-F-5-methyl-2'-deoxy-UTP, 2'-OMe- 5-Me-UTP, 2'-OMe-pseudo-UTP, 2'-α-ethynyluridine TP, 2'-α-trifluoromethyluridine TP, 2'-β-ethynyluridine TP, 2'-β-trifluoromethyluridine TP, 2'-deoxy-2',2'-difluorouridine TP, 2'-deoxy-2'-α-mercaptouridine TP, 2'-deoxy-2'-α-thiomethoxyuridine TP, 2'-deoxy-2'-β-aminouridine TP, 2'-deoxy-2'-β-azidouridine TP, 2'-deoxy-2'-β-bromouridine TP, 2'-deoxy Oxy-2'-b-chlorouridine TP, 2'-deoxy-2'-b-fluorouridine TP, 2'-deoxy-2'-b-iodouridine TP, 2'-deoxy-2'-b-mercaptouridine TP, 2'-deoxy-2'-b-thiomethoxyuridine TP, 2-methoxy-4-thiouridine, 2-methoxyuridine, 2'-O-methyl-5-(1-propynyl)uridine TP, 3-alkyl-pseudo-UTP, 4'-azidouridine TP, 4'-carbocyclic uridine TP, 4'-ethynyluridine TP, 5-(1-propynyl)ara-uridine TP,5-(2-furanyl)uridine TP, 5-cyanouridine TP, 5-dimethylaminouridine TP, 5'-homouridine TP, 5-iodo-2'-fluoro-deoxyuridine TP, 5-phenylethynyluridine TP, 5-trideuteromethyl-6-deuterouridine TP, 5-trifluoromethyluridine TP, 5-vinylaruridine TP, 6-(2,2,2-trifluoroethyl)-pseudo-UTP, 6-(4-morpholino)-pseudo-UTP, 6-(4-thiomorpholino)-pseudo-UTP, 6-(substituted phenyl)-pseudo-UTP P, 6-amino-pseudo-UTP, 6-azido-pseudo-UTP, 6-bromo-pseudo-UTP, 6-butyl-pseudo-UTP, 6-chloro-pseudo-UTP, 6-cyano-pseudo-UTP, 6-dimethylamino-pseudo-UTP, 6-ethoxy-pseudo-UTP, 6-ethylcarboxylate-pseudo-UTP, 6-ethyl-pseudo-UTP, 6-fluoro-pseudo-UTP, 6-formyl-pseudo-UTP, 6-hydroxyamino-pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP P, 6-iso-propyl-pseudo-UTP, 6-methoxy-pseudo-UTP, 6-methylamino-pseudo-UTP, 6-methyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-propyl-pseudo-UTP, 6-tert-butyl-pseudo-UTP, 6-trifluoromethoxy-pseudo-UTP, 6-trifluoromethyl-pseudo-UTP, alpha-thio-pseudo-UTP, pseudouridine 1-(4-methylbenzenesulfonate)TP, pseudouridine 1-(4-methylbenzoate) )TP, pseudouridine TP 1-[3-(2-ethoxy)]propionic acid, pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid,Pseudouridine TP1-methylphosphonate, Pseudouridine TP1-methylphosphonate diethyl ester, Pseudo-UTP-N1-3-propionic acid, Pseudo-UTP-N1-4-butanoic acid, Pseudo-UTP-N1-5-pentanoic acid, Pseudo-UTP-N1-6-hexanoic acid, Pseudo-UTP-N1-7-heptanoic acid, Pseudo-UTP-N1-methyl-p-benzoic acid, Pseudo-UTP-N1-p-benzoic acid, Wybutosin, Hydroxywybutosin, Isowyosin, Peroxywybutosin, Undermodified Hydroxywybutosin, 4-demethylwybutosin, 2,6-(diamino)purine, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenothiazin-l-yl, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, 2(amino)purine, 2,4,5-(trimethyl)phenyl, 2'methyl, 2'amino, 2'azido, 2'fluro-cytidine, 2'methyl, 2'amino, 2'azido, 2'fluro-adenine , 2'methyl, 2'amino, 2'azido, 2'fluro-uridine, 2'-amino-2'-deoxyribose, 2-amino-6-chloro-purine, 2-aza-inosinyl, 2'-azido-2'-deoxyribose, 2'fluoro-2'-deoxyribose, 2'-fluoro-modified base, 2'-O-methyl-ribose, 2-oxo-7-aminopyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, 2-pyridinone, 3-nitropyrrole, 3-(methyl)-7-(propynyl)isocarbostyril, 3-(methyl)isocarbostyril, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, 5-nitroindole, 5-substituted pyrimidines, 5-(methyl)isocarbostyrilyl, 5-nitroindole, 6-(aza)pyrimidine, 6-(azo)thymine, 6-(methyl)-7-(aza)indolyl, 6-chloro-purine, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aza)indolyl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio) -3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, Dimethylaminomethyl 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(propynyl)isocarbostyril, 7-(propynyl)isocarbostyril, propynyl-7-(aza)indolyl, 7-deaza-inosinyl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 9-(methyl)-imidizopyridinyl, aminoindolyl, anthracenyl, bis-ortho-(aminoalkylhydroxy)- (ortho-substituted)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, difluorotolyl, hypoxanthine, imidizopyridinyl, inosinyl, isocarbostyrilyl, isoguanisine, N2-substituted purines, N6-methyl-2-amino-purines, N6-substituted purines, N-alkylated derivatives, naphthalenyl, nitrobenzimidazolyl, nitroimidazolyl, nitroindazolyl, nitropyrazolyl, nubularine, O6-substituted purines, O-alkylated derivatives,ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, oxoformycin TP, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pentacenyl, phenanthracenyl, phenyl, , propynyl-7-(aza)indolyl, pyrenyl, pyridopyrimidin-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, pyrrolo-pyrimidin-2-one-3-yl, pyrrolopyrimidinyl, pyrrolopyridinyl, stilbenzyl, substituted 1,2,4-triazoles, tetracenyl, tubercidin, xanthine, xanthosine-5'-TP, 2-thio-zebularine, 5-aza-2-thio-zebularine , 7-deaza-2-amino-purine, pyridin-4-one ribonucleoside, 2-amino-riboside-TP, formycin ATP, formycin BTP, pyrrolosine TP, 2'-OH-ara-adenosine TP, 2'-OH-ara-cytidine TP, 2'-OH-ara-uridine TP, 2'-OH-ara-guanosine TP, 5-(2-carbomethoxyvinyl)uridine TP, and N6-(19-amino-pentaoxanone adecyl)adenosine TP.

[0167] In some embodiments, a polynucleotide (eg, an RNA polynucleotide such as an mRNA polynucleotide) comprises a combination of at least two (eg, two, three, four or more) of the foregoing modified nucleobases.

[0168] In some embodiments, the modified nucleobase in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) is pseudouridine (ψ), 2-thiouridine (s2U), 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- Dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-meth cytidine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4'-thiouridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A) and 2,6-diaminopurine, (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7- Deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-lysidine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, Selected from the group consisting of 7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queosine, methylated hypomodified hydroxywyosine, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Q base, preQ0 base, preQ1 base, and combinations of two or more thereof. In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, a polyribonucleotide (e.g., an RNA polyribonucleotide, such as an mRNA polyribonucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases. In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases.

[0169] In some embodiments, modified nucleobases in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) are selected from the group consisting of 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, a polyribonucleotide comprises a combination of at least two (e.g., two, three, four, or more) of the foregoing modified nucleobases, including, but not limited to, chemical modifications.

[0170] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (mC). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-methyl-pseudouridine (mψ). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-ethyl-pseudouridine (eψ). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-methyl-pseudouridine (mψ) and 5-methyl-cytidine (mC). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 1-ethyl-pseudouridine (eψ) and 5-methyl-cytidine (mC). In some embodiments, a polyribonucleotide (e.g., an RNA such as an mRNA) comprises 2-thiouridine (sU). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise methoxy-uridine (mo5U). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 2'-O-methyluridine. In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, polyribonucleotides (e.g., RNA such as mRNA) comprise N6-methyl-adenosine (m6A). In some embodiments, the polyribonucleotide (eg, RNA such as mRNA) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0171] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., completely modified, modified throughout the entire sequence) with a particular modification. For example, a polynucleotide can be uniformly modified with 1-methyl-pseudouridine, i.e., all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a polynucleotide can be uniformly modified by replacing any type of nucleoside residue present in the sequence with a modified residue, such as those described above.

[0172] Exemplary nucleobases and nucleosides having modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0173] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having modified uridines include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxyuridine, 2-thiouridine, 5-cyanouridine, 2'-O-methyluridine, and 4'-thiouridine.

[0174] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0175] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine.

[0176] The polynucleotide of the present disclosure can be partially or completely modified throughout the entire length of the molecule.For example, one or more or all or a given type of nucleotide (for example, purine or pyrimidine, or any one or more or all of A, G, U, C) in the polynucleotide of the present disclosure or in a given sequence region thereof (for example, mRNA with or without poly-A tail) can be uniformly modified.In some embodiments, all nucleotides X in the polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, where X is any one of the nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0177] Polynucleotides may contain modified nucleotides from about 1% to about 100% (relative to the overall nucleotide content, or relative to one or more types of nucleotides (i.e., any one or more of A, G, U, or C)), or any percentage in between (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-95%, 10%-10 ... It may contain up to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%. It will be understood that any remaining percentage will be accounted for by unmodified A, G, U or C occurrences.

[0178] A polynucleotide can contain as little as 1% and as much as 100% modified nucleotides, or any percentage therebetween, e.g., at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, a polynucleotide can contain modified pyrimidines such as modified uracil or modified cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in a polynucleotide are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils can be replaced by a compound with a single unique structure, or by multiple compounds with different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in a polynucleotide are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines may be replaced by a compound having a single unique structure or by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0179] Thus, in some embodiments, the RNA vaccine comprises 5'UTR elements, optionally a codon-optimized open reading frame and 3'UTR elements, a poly(A) sequence and / or a polyadenylation signal, and the RNA is not chemically modified.

[0180] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-hydroxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5Um), 3,2'-O-dimethyl-uridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine and 5-[3-(1-E-propenylamino)]uridine.

[0181] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0182] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6A), N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine and N6-(19-amino-pentaoxanone adecyl)-adenosine.

[0183] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m), 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OhyW), and low-modified hydroxywyobutosine (OhyW). * ), 7-deaza-guanosine, queosine (Q), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G +), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0184] In vitro transcription of RNA (e.g., mRNA) The VZV vaccines of the present disclosure include at least one RNA polynucleotide, such as an mRNA (e.g., a modified mRNA). The mRNA is transcribed in vitro, for example, from a template DNA referred to as an "in vitro transcription template." In some embodiments, the at least one RNA polynucleotide has at least one chemical modification. The at least one chemical modification can include, but is not expressly limited to, any of the modifications described herein.

[0185] In vitro transcription of RNA is known in the art and is described in International Publication No. WO / 2014 / 152027, which is incorporated herein by reference in its entirety. For example, in some embodiments, RNA transcripts are generated using an unamplified linear DNA template in an in vitro transcription reaction to produce the RNA transcript. In some embodiments, the RNA transcript is capped by enzymatic capping. In some embodiments, the RNA transcript is purified by chromatography, e.g., using an oligo-dT substrate. Some embodiments exclude the use of DNase. In some embodiments, the RNA transcript is synthesized from an unamplified linear DNA template encoding a gene of interest by an enzymatic in vitro transcription reaction utilizing T7 phage RNA polymerase and a desired chemical nucleotide triphosphate. Any number of RNA polymerases or variants can be used in the methods of the invention. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or a mutant polymerase (e.g., a polymerase capable of incorporating modified nucleic acids and / or nucleotides, including, but not limited to, chemically modified nucleic acids and / or nucleotides).

[0186] In some embodiments, unamplified linearized plasmid DNA is used as template DNA for in vitro transcription. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA polynucleotide, such as, but not limited to, VZV RNA, e.g., VZV mRNA. In some embodiments, the cell is, for example, a bacterial cell, e.g., E. coli, e.g., DH-1 cells, transfected with a plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template comprises an RNA polymerase promoter, e.g., a T7 promoter, operably linked 5' to a gene of interest.

[0187] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a poly A tail. The composition and length of the particular nucleic acid sequence of the in vitro transcription template depends on the mRNA encoded by the template.

[0188] "5' untranslated region" (UTR) refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of the mRNA transcript that is translated by the ribosome) that does not encode a polypeptide.

[0189] "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (i.e., 3') from a stop codon (i.e., a codon in an mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0190] An "open reading frame" is a contiguous stretch of DNA that begins with a start codon (eg, methionine (ATG)), ends with a stop codon (eg, TAA, TAG, or TGA), and encodes a polypeptide.

[0191] A "poly-A tail" is a region of an mRNA downstream, e.g., directly downstream (i.e., 3') from the 3' UTR, that contains a large number of consecutive adenosine monophosphates. A poly-A tail can contain 10 to 300 adenosine monophosphates. For example, a poly-A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains 50 to 250 adenosine monophosphates. Within relevant biological environments (e.g., within cells, in vivo), the poly(A) tail functions, for example, to protect the mRNA from enzymatic degradation in the cytoplasm and also assists in transcription termination and / or transport and translation of the mRNA from the nucleus.

[0192] In some embodiments, the polynucleotide comprises 200 to 3,000 nucleotides. For example, the polynucleotide may comprise 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides.

[0193] Treatment method Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of VZV in humans and other animals. VZV RNA vaccines can be used as therapeutic or prophylactic agents. They can be used as drugs for the prevention and / or treatment of infectious diseases. In exemplary embodiments, the VZV RNA vaccines of the present invention are used to provide prophylactic protection against chickenpox and herpes zoster. Chickenpox is an acute infectious disease caused by VZV. Primary infection with the varicella-zoster virus, which results in chickenpox (varicella), can also lead to complications, including viral or secondary bacterial pneumonia. Even after the clinical symptoms of chickenpox have resolved, VZV remains latent in the nervous system of infected individuals in the trigeminal and dorsal root ganglia. It can become activated later in life and return to the skin from sensory ganglia, where it can cause the disease (rash) known as shingles or herpes zoster, and can also cause several neurological conditions ranging from aseptic meningitis to encephalitis. The VZV vaccines of the present disclosure can be used to prevent and / or treat both primary infection (chickenpox) and reactivated viral infection (shingles or herpes zoster), and may be particularly useful for the prevention and / or treatment of herpes zoster in immunocompromised and elderly patients to prevent or reduce the severity and / or duration of herpes zoster.

[0194] Prophylactic protection from VZV can be achieved after administration of the VZV RNA vaccine of the present disclosure. The vaccine can be administered once, twice, three times, four times, or more times, although a single administration of the vaccine (optionally followed by one booster) may be sufficient. Although less desirable, it is possible to administer the vaccine to infected individuals to achieve a therapeutic response. Dosages may need to be adjusted accordingly.

[0195] In one embodiment of the present disclosure, a method for eliciting an immune response against VZV in a subject is provided. The method involves administering to the subject a VZV RNA vaccine comprising at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof, thereby inducing an immune response in the subject specific to the VZV antigenic polypeptide or immunogenic fragment thereof, and the subject's anti-antigenic polypeptide antibody titer is increased after vaccination compared to the anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV. An "anti-antigenic polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide.

[0196] A prophylactically effective dose is a therapeutically effective dose that prevents viral infection at a clinically acceptable level. In some embodiments, a therapeutically effective dose is the dose listed in the vaccine package insert. As used herein, a conventional vaccine refers to a vaccine other than the mRNA vaccine of the present disclosure. For example, conventional vaccines include, but are not limited to, live microbial vaccines, killed microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, VLP vaccines, etc. In an exemplary embodiment, a conventional vaccine is a vaccine that has received regulatory approval and / or been registered by a national drug regulatory agency, such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0197] In one embodiment of the present invention, a method for eliciting an immune response against VZV in a subject is provided. The method involves administering to the subject a VZV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof, thereby inducing an immune response in the subject specific to the VZV antigenic polypeptide or immunogenic fragment thereof, and the subject's anti-antigenic polypeptide antibody titer is increased after vaccination compared to the anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV. An "anti-antigenic polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide.

[0198] A prophylactically effective dose is a therapeutically effective dose that prevents viral infection at a clinically acceptable level. In some embodiments, a therapeutically effective dose is the dose listed in the package insert of the vaccine. As used herein, a conventional vaccine refers to a vaccine other than the mRNA vaccine of the present invention. For example, conventional vaccines include, but are not limited to, live microbial vaccines, killed microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, etc.

[0199] In some embodiments, the subject's anti-antigenic polypeptide antibody titer increases by 1 log to 10 logs after vaccination compared to the anti-antigenic polypeptide antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV.

[0200] In some embodiments, the subject's anti-antigenic polypeptide antibody titer increases by 1 log after vaccination compared to the anti-antigenic polypeptide antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV.

[0201] In some embodiments, the subject's anti-antigenic polypeptide antibody titer increases by 2 logs after vaccination compared to the anti-antigenic polypeptide antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV.

[0202] In some embodiments, the subject's anti-antigenic polypeptide antibody titer increases by 3 logs after vaccination compared to the anti-antigenic polypeptide antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV.

[0203] In some embodiments, the subject's anti-antigenic polypeptide antibody titer increases by 5 logs after vaccination compared to the anti-antigenic polypeptide antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV.

[0204] In some embodiments, the subject's anti-antigenic polypeptide antibody titer increases by 10 logs after vaccination compared to the anti-antigenic polypeptide antibody titer of a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV.

[0205] In another embodiment of the present invention, there is provided a method for eliciting an immune response against VZV in a subject, comprising administering to the subject a VZV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the VZV antigenic polypeptide or immunogenic fragment thereof, wherein the immune response in the subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine against VZV at a dose level 2- to 100-fold higher than that of the RNA vaccine.

[0206] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at twice the dose level as compared to a VZV RNA vaccine.

[0207] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a dose level three times higher than that of a VZV RNA vaccine.

[0208] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a four-fold dose level relative to a VZV RNA vaccine.

[0209] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a 5-fold dose level relative to a VZV RNA vaccine.

[0210] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a 10-fold higher dose level than a VZV RNA vaccine.

[0211] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a 50-fold higher dose level than a VZV RNA vaccine.

[0212] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a 100-fold higher dose level than a VZV RNA vaccine.

[0213] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a dose level 10-1000 times higher than that of a VZV RNA vaccine.

[0214] In some embodiments, the subject's immune response is equivalent to the immune response of a subject vaccinated with a conventional vaccine at a dose level 100-1000 times higher than that of a VZV RNA vaccine.

[0215] In other embodiments, the immune response is assessed by determining the [protein] antibody titer in the subject.

[0216] In another aspect, the present invention provides a method for inducing an immune response against VZV in a subject by administering to the subject a VZV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide or immunogenic fragment thereof, thereby inducing an immune response in the subject specific to the VZV antigenic polypeptide or immunogenic fragment thereof, wherein the immune response in the subject is induced 2 days to 10 weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against VZV. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a conventional vaccine that is 2-fold to 100-fold higher than the dose of the RNA vaccine.

[0217] In some embodiments, an immune response in a subject is induced two days earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0218] In some embodiments, an immune response in a subject is induced three days earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0219] In some embodiments, an immune response in a subject is induced one week earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0220] In some embodiments, an immune response in a subject is induced two weeks earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0221] In some embodiments, an immune response in a subject is induced three weeks earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0222] In some embodiments, an immune response in a subject is induced 5 weeks earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0223] In some embodiments, an immune response in a subject is induced 10 weeks earlier than the immune response elicited in a subject vaccinated with a prophylactically effective dose of a conventional vaccine.

[0224] A method for inducing an immune response against VZV in a subject by administering to the subject a VZV RNA vaccine having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not contain a stabilizing element and the vaccine is not co-formulated with or co-administered with an adjuvant.

[0225] Broad-spectrum VZV vaccine Situations are envisioned in which there is a risk of infection with two or more VZV strains. RNA (e.g., mRNA) therapeutic vaccines are particularly suited to combination vaccine approaches due to several factors, including, but not limited to, speed of production and the ability to rapidly tailor vaccines to accommodate recognized geographic threats. Furthermore, because such vaccines utilize the human body to produce antigenic proteins, they are suitable for producing larger, more complex antigenic proteins, allowing for proper folding, surface expression, antigen presentation, etc. in human subjects. To protect against two or more VZV strains, combination vaccines can be administered that contain RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first VZV and further contain RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second VZV. The RNAs (mRNAs) can be formulated together, for example, in a single lipid nanoparticle (LNP) or in separate LNPs intended for co-administration.

[0226] Flagellin adjuvant Flagellin is a monomeric protein of approximately 500 amino acids that polymerizes to form flagella, which are involved in bacterial movement. Flagellin is expressed by various flagellated bacteria (e.g., Salmonella typhimurium) and amastigotes (e.g., Escherichia coli). Flagellin detection by innate immune cells (e.g., dendritic cells, macrophages) is mediated by Toll-like receptor 5 (TLR5) and Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and NLRs have been identified to play a role in activating innate and adaptive immune responses. Therefore, flagellin provides an adjuvant effect in vaccines.

[0227] Nucleotide and amino acid sequences encoding known flagellin polypeptides are publicly available in the NCBI GenBank database, including flagellin sequences from S. Typhimurium, H. pylori, V. Cholera, S. marcesens, S. flexneri, T. pallidum, L. pneumophila, B. burgdorferei, C. difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P. mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli, among others.

[0228] As used herein, flagellin polypeptide refers to full-length flagellin protein, immunogenic fragments thereof, and peptides having at least 50% sequence identity to the flagellin protein or immunogenic fragments thereof. Exemplary flagellin proteins include flagellins from Salmonella typhi (UniPro entry number Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonella choleraesuis (Q6V2X8) and SEQ ID NOs: 115-117. In some embodiments, the flagellin polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the flagellin protein or immunogenic fragments thereof.

[0229] In some embodiments, the flagellin polypeptide is an immunogenic fragment. An immunogenic fragment is a portion of the flagellin protein that elicits an immune response. In some embodiments, the immune response is a TLR5 immune response. An example of an immunogenic fragment is a flagellin protein in which all or part of the hinge region is deleted or replaced with other amino acids. For example, an antigenic polypeptide can be inserted into the hinge region. The hinge region is the hypervariable region of flagellin. The hinge region of flagellin is also referred to as the "D3 domain or region," "propeller domain or region," "hypervariable domain or region," and "variable domain or region." As used herein, "at least a portion of the hinge region" refers to any portion of the hinge region or the entire hinge region of flagellin. In other embodiments, the immunogenic fragment of flagellin is a 20-, 25-, 30-, 35-, or 40-amino acid C-terminal fragment of flagellin.

[0230] The flagellin monomer is formed by domains D0 to D3. The stem, D0 and D1, consist of a long tandem alpha helix and are highly conserved among various bacteria. The D1 domain contains several amino acid stretches useful for TLR5 activation. The entire D1 domain or one or more active regions within this domain are immunogenic fragments of flagellin. Examples of immunogenic regions within the D1 domain include residues 88-114 and 411-431 of Salmonella typhimurium FliC flagellin. Of the 13 amino acids in the 88-100 region, at least six substitutions are possible between Salmonella flagellin and other flagellin proteins while retaining TLR5 activation. Thus, immunogenic fragments of flagellin contain a flagellin-like sequence that activates TLR5 and contains a 13-amino acid motif that is 53% or more identical to the Salmonella FliC 88-100 sequence (LQRVRELAVQSAN, SEQ ID NO: 118).

[0231] In some embodiments, RNA (e.g., mRNA) vaccines comprise RNA encoding a fusion protein of flagellin and one or more antigenic polypeptides. As used herein, "fusion protein" refers to the linkage of two components of a construct. In some embodiments, the carboxy terminus of an antigenic polypeptide is fused or linked to the amino terminus of a flagellin polypeptide. In other embodiments, the amino terminus of an antigenic polypeptide is fused or linked to the carboxy terminus of a flagellin polypeptide. A fusion protein can include, for example, 1, 2, 3, 4, 5, 6, or more flagellin polypeptides linked to 1, 2, 3, 4, 5, 6, or more antigenic polypeptides. When two or more flagellin polypeptides and / or two or more antigenic polypeptides are linked, such constructs may be referred to as "multimers."

[0232] The components of the fusion protein may be directly linked to each other or may be connected via a linker. For example, the linker may be an amino acid linker. The amino acid linker for linking the components of the fusion protein encoded by an RNA (e.g., mRNA) vaccine may include, for example, at least one element selected from the group consisting of a lysine residue, a glutamic acid residue, a serine residue, and an arginine residue. In some embodiments, the linker is 1 to 30, 1 to 25, 1 to 25, 5 to 10, 5, 15, or 5 to 20 amino acids in length.

[0233] In other embodiments, the RNA (e.g., mRNA) vaccine comprises at least two separate RNA polynucleotides, one encoding one or more antigenic polypeptides and the other encoding a flagellin polypeptide, which may be formulated together in a carrier such as a lipid nanoparticle.

[0234] Therapeutic and Prophylactic Compositions For example, provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, and / or diagnosis of VZV in humans and other animals. VZV RNA (e.g., mRNA) vaccines can be used as therapeutic or prophylactic agents. They can be used as agents for the prevention and / or treatment of infectious diseases. In some embodiments, the VZV vaccines of the present invention can be envisioned for use in priming immune effector cells, e.g., by ex vivo activation of peripheral blood mononuclear cells (PBMCs) followed by infusion (reinfusion) into a subject.

[0235] In an exemplary embodiment, a VZV vaccine containing an RNA polynucleotide described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

[0236] A VZV RNA vaccine can induce translation of a polypeptide (e.g., an antigen or immunogen) in a cell, tissue, or organism. In exemplary embodiments, such translation occurs in vivo, although embodiments in which such translation occurs ex vivo, in culture, or in vitro are also contemplated. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of a composition containing a VZV RNA vaccine that contains a polynucleotide having at least one translatable region encoding an antigenic polypeptide.

[0237] An "effective amount" of a VZV RNA vaccine is provided based, at least in part, on the target tissue, target cell type, administration method, physical characteristics of the polynucleotide (e.g., size and degree of modified nucleosides), and other components of the VZV RNA vaccine, as well as other determinants. Generally, an effective amount of a VZV RNA vaccine composition will result in an induced or boosted immune response correlated with antigen production in cells. In general, an effective amount of a VZV RNA vaccine containing an RNA polynucleotide with at least one chemical modification is preferably more effective than a composition containing a similar unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production can be indicated by increased cell transfection (percentage of cells transfected with the RNA vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., indicated by a longer protein translation period from the modified polynucleotide), or a change in the antigen-specific immune response of host cells.

[0238] The term "pharmaceutical composition" refers to a combination of an active ingredient with an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic applications. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects after or upon administration to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense of being compatible with the active ingredient and capable of stabilizing the active ingredient. One or more solubilizing agents may be utilized as pharmaceutical carriers for delivery of the active ingredient. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents that achieve a usable composition in dosage form. Other examples of carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical requirements for their use, are described in Remington's Pharmaceutical Sciences.

[0239] In some embodiments, an RNA vaccine (including a polynucleotide and its encoded polypeptide) according to the present disclosure can be used for the treatment or prevention of VZV.

[0240] VZV RNA vaccines can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization scheme, or early in infection, during incubation or post-symptomatic active infection. In some embodiments, the amount of the RNA vaccine of the present disclosure provided to a cell, tissue, or subject can be an amount effective for immunoprophylaxis.

[0241] VZV RNA (e.g., mRNA) vaccines can be administered together with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound can be an adjuvant or booster. When used herein with respect to a prophylactic composition such as a vaccine, the term "booster" refers to an additional administration of a prophylactic (vaccine) composition. A booster (or booster vaccine) can be administered after a previous administration of a prophylactic composition. The time from the first administration of the prophylactic composition to the booster administration may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, The period may be 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or greater than 99 years. In exemplary embodiments, the time from the first administration of the prophylactic composition to the booster administration may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0242] In some embodiments, VZV RNA vaccines can be administered intramuscularly, intranasally, or intradermally, similar to the administration of inactivated vaccines known in the art.

[0243] VZV RNA vaccines can be used in a variety of settings depending on the prevalence of infection or the extent or level of unmet medical need. As a non-limiting example, RNA vaccines can be used to treat and / or prevent a variety of infectious diseases. RNA vaccines have superior properties compared to commonly available antivirals in that they produce greater antibody titers and a faster response.

[0244] Provided herein are pharmaceutical compositions comprising VZV RNA vaccines and RNA vaccine compositions and / or complexes, optionally in combination with one or more pharmaceutically acceptable excipients.

[0245] VZV RNA (e.g., mRNA) vaccines can be formulated or administered alone or together with one or more other components. For example, a VZV RNA vaccine (vaccine composition) can include other components, including, but not limited to, an adjuvant.

[0246] In some embodiments, the VZV RNA vaccine does not include an adjuvant (is adjuvant-free).

[0247] VZV RNA (e.g., mRNA) vaccines can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, vaccine compositions include at least one additional active agent, such as a therapeutically active agent, a prophylactically active agent, or a combination of both. Vaccine compositions can be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceuticals, such as vaccine compositions, can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, 2005, incorporated herein by reference in its entirety.

[0248] In some embodiments, the VZV RNA vaccine is administered to a human, human patient, or subject. For purposes of this disclosure, the term "active ingredient" generally refers to an RNA vaccine or a polynucleotide contained therein, such as an RNA polynucleotide (e.g., an mRNA polynucleotide) that encodes an antigenic polypeptide.

[0249] Formulations of the vaccine compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods include the step of bringing into association the active ingredient (e.g., mRNA polynucleotide) with an excipient and / or one or more other accessory ingredients, and then, as necessary and / or desirable, dividing, shaping, and / or packaging the product into the desired single or multi-dose units.

[0250] The relative amounts of active ingredient, pharmaceutically acceptable excipient and / or optional ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the characteristics, size and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may comprise from 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0251] VZV RNA vaccines can be formulated with one or more excipients that (1) enhance stability, (2) increase cell transfection, (3) enable sustained or delayed release (e.g., from a depot formulation), (4) alter biodistribution (e.g., targeting to specific tissues or cell types), (5) increase in vivo translation of the encoded protein, and / or (6) alter the in vivo release profile of the encoded protein (antigen). Excipients can include any and all of conventional excipients, such as solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, tonicity agents, thickeners or emulsifiers, and preservatives, as well as, but not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the VZV RNA vaccine (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0252] stabilizing element Natural eukaryotic mRNA molecules are known to contain stabilizing elements, such as, but not limited to, untranslated regions (UTRs) at their 5' ends (5'UTRs) and / or UTRs at their 3' ends (3'UTRs), in addition to other structural features such as a 5' cap structure or a 3' poly(A) tail. Both the 5'UTR and 3'UTR are typically transcribed from genomic DNA and are components of premature mRNAs. Characteristic structural features of mature mRNAs, such as the 5' cap and 3' poly(A) tail, are usually added to transcribed (premature) mRNAs during mRNA processing. The 3' poly(A) tail is typically a stretch of adenine nucleotides added to the 3' end of a transcribed mRNA and can contain up to approximately 400 adenine nucleotides. In some embodiments, the length of the 3' poly(A) tail may be an essential factor for the stability of an individual mRNA.

[0253] In some embodiments, the RNA vaccine may contain one or more stabilizing elements. The stabilizing element may include, for example, a histone stem loop. A 32-kDa protein, stem-loop binding protein (SLBP), has been identified. SLBP associates with histone stem loops at the 3' end of histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle, peaking during S phase, when histone mRNA levels also increase. This protein has been shown to be required for efficient 3'-end processing of histone pre-mRNA by U7 snRNP. SLBP continues to associate with stem loops after processing and then stimulates translation of mature histone mRNA into histone proteins in the cytoplasm. The RNA-binding domain of SLBP is conserved across metazoans and protozoans, and its binding to histone stem loops depends on the loop structure. The minimal binding site is at least three nucleotides 5' and two nucleotides 3' to the stem loop.

[0254] In some embodiments, the RNA vaccine comprises a coding region, at least one histone stem loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally enhances the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selection protein (e.g., α-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0255] In some embodiments, the combination of a poly(A) sequence or a polyadenylation signal with at least one histone stem-loop, both of which represent alternative mechanisms in nature, acts synergistically to increase protein expression above levels observed with either of the individual elements. The synergistic effect of the combination of poly(A) and at least one histone stem-loop has been found to be independent of the order of the elements or the length of the poly(A) sequence.

[0256] In some embodiments, the RNA vaccine does not contain a histone downstream element (HDE). A "histone downstream element" (HDE) comprises a purine-rich polynucleotide stretch of approximately 15-20 nucleotides 3' to the natural stem-loop, which represents a binding site for U7 snRNA, which is involved in processing histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not contain an intron.

[0257] In some embodiments, RNA vaccines may or may not contain enhancer and / or promoter sequences, which may be modified or unmodified, activated or inactivated. In some embodiments, histone stem-loops typically originate from histone genes and involve intramolecular base pairing of two adjacent, partially or completely reverse-complementary sequences separated by a short spacer, forming the loop of the structure. The unpaired loop region typically cannot base pair with either of the stem-loop elements. This occurs more frequently in RNA because it is an important component of many RNA secondary structures, but can also exist in single-stranded DNA. The stability of the stem-loop structure typically depends on the length of the paired region, the number of mismatches or bulges, and the base composition. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) can occur. In some embodiments, at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0258] In other embodiments, the RNA vaccine may be depleted of one or more AU-rich sequences. These sequences are sometimes called AURES, and are destabilizing sequences found in the 3'UTR. AURES may be removed from the RNA vaccine. Alternatively, AURES may be left in the RNA vaccine.

[0259] Nanoparticle formation In some embodiments, the VZV RNA (e.g., mRNA) vaccine is formulated in nanoparticles. In some embodiments, the VZV RNA vaccine is formulated in lipid nanoparticles. In some embodiments, the VZV RNA vaccine is formulated in lipid-polycation complexes called cationic lipid nanoparticles. Formation of lipid nanoparticles can be achieved by methods known in the art and / or described in U.S. Patent Application Publication No. 20120178702 (incorporated herein by reference in its entirety). By way of non-limiting example, polycations can include cationic peptides or polypeptides, including, but not limited to, polylysine, polyornithine, and / or polyarginine, and the cationic peptides described in International Publication No. WO2012013326 or U.S. Patent Application Publication No. US20130142818 (incorporated herein by reference in their entirety). In some embodiments, the VZV RNA vaccine is formulated in lipid nanoparticles comprising a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0260] Lipid nanoparticle formulation can be influenced by biophysical parameters, including but not limited to, the selection of cationic lipid components, the degree of saturation of cationic lipids, the nature of PEGylation, the ratio of all components, and size.In one example by Semple et al. (Nature Biotech.2010 28:172-176, the entirety of which is incorporated herein by reference), the lipid nanoparticle formulation is composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.In another example, it has been shown that changing the composition of cationic lipids can more efficiently deliver siRNA to various antigen-presenting cells (Basha et al.Mol Ther.2011 19:2186-2200, the entirety of which is incorporated herein by reference).

[0261] In some embodiments, the lipid nanoparticle formulation may comprise 35-45% cationic lipid, 40-50% cationic lipid, 50-60% cationic lipid, and / or 55-65% cationic lipid. In some embodiments, the lipid to RNA (e.g., mRNA) ratio in the lipid nanoparticle may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, and / or at least 30:1.

[0262] In some embodiments, the ratio of PEG in the lipid nanoparticle formulation may be increased or decreased, and / or the carbon chain length of the PEG lipid may be changed from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, the lipid nanoparticle formulation may contain PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) at a lipid molar ratio of 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0%, and / or 3.0% to 6.0% relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, PEG-c-DOMG may be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA.

[0263] In some embodiments, the VZV RNA (e.g., mRNA) vaccine formulation is a nanoparticle comprising at least one lipid, which may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530), PEGylated lipids, and aminoalcohol lipids.

[0264] In some embodiments, the lipid is [ka] is.

[0265] In some embodiments, the lipid is [ka] (L530).

[0266] In some embodiments, the lipids may be cationic lipids such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol cationic lipids, which may be lipids described in U.S. Patent Application Publication No. US20130150625 (incorporated herein by reference in its entirety) and / or may be made by the methods described therein. Non-limiting examples of cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 of US20130150625), 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 of US20130150625), 2-amino-3-[ (9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 of US20130150625), and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 of US20130150625), or any pharmaceutically acceptable salt or stereoisomer thereof.

[0267] Lipid nanoparticle formulations typically contain lipids, in particular ionic cationic lipids such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further contain a neutral lipid sterol and a molecule capable of reducing particle aggregation, such as a PEG lipid or a PEG-modified lipid.

[0268] In some embodiments, the lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol:0.5-15% PEG lipid.

[0269] In some embodiments, the lipid nanoparticle formulation comprises 25% to 75% on a molar basis, e.g., 35 to 65%, 45 to 65%, 60%, 57.5%, 50%, or 40% on a molar basis, of a cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0270] In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 15% on a molar basis, e.g., 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% neutral lipid on a molar basis. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises 5% to 50% on a molar basis (e.g., 15% to 45%, 20% to 40%, 40%, 38.5%, 35%, or 31% on a molar basis) of a sterol. A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 20% (e.g., 0.5-10%, 0.5-5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis) PEG-lipids or PEG-modified lipids. In some embodiments, the PEG-lipids or PEG-modified lipids comprise PEG molecules with an average molecular weight of 2,000 Da. In some embodiments, the PEG-lipids or PEG-modified lipids comprise PEG molecules with an average molecular weight of less than 2,000, e.g., about 1,500 Da, about 1,000 Da, or about 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG) and PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), the entire contents of which are incorporated herein by reference).

[0271] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 25-75% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 0.5-15% neutral lipid; 5-50% sterol; and 0.5-20% PEG lipid or PEG-modified lipid.

[0272] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 35-65% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% neutral lipid, 15-45% sterol, and 0.5-10% PEG lipid or PEG-modified lipid.

[0273] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 45-65% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% neutral lipid, 25-40% sterol, and 0.5-10% PEG lipid or PEG-modified lipid.

[0274] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 60% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% neutral lipid, 31% sterol, and 1.5% PEG lipid or PEG-modified lipid.

[0275] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% neutral lipid, 38.5% sterol, and 1.5% PEG lipid or PEG-modified lipid.

[0276] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% neutral lipid, 35% sterol, 4.5% or 5% PEG lipid or PEG-modified lipid, and 0.5% targeting lipid.

[0277] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 40% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% neutral lipid, 40% sterol, and 5% PEG lipid or PEG-modified lipid.

[0278] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.2% cationic lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% neutral lipid, 34.3% sterol, and 1.4% PEG lipid or PEG-modified lipid.

[0279] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.5% cationic lipid selected from PEG lipid, PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the entire contents of which are incorporated herein by reference), 7.5% neutral lipid, 31.5% sterol, and 3.5% PEG lipid or PEG-modified lipid.

[0280] In some embodiments, the lipid nanoparticle formulation consists essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation consists essentially of a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

[0281] In some embodiments, the lipid molar ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DMG, PEG-DSG, or PEG-DPG)), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid (e.g., DPPC) / Chol / PEG-modified lipid (e.g., PEG-cDMA)), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DMG)), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DSG)), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DMG)), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid (e.g., DSPC) / Chol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA).

[0282] Non-limiting examples of lipid nanoparticle compositions and methods for making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533, and Maier et al. (2013) Molecular Therapy 21, 1570-1578, the contents of each of which are incorporated herein by reference in their entirety.

[0283] In some embodiments, lipid nanoparticle formulations may comprise cationic lipids, PEG lipids, and structured lipids, and optionally, non-cationic lipids. As a non-limiting example, lipid nanoparticles may comprise 40-60% cationic lipids, 5-15% non-cationic lipids, 1-2% PEG lipids, and 30-50% structured lipids. As another non-limiting example, lipid nanoparticles may comprise 50% cationic lipids, 10% non-cationic lipids, 1.5% PEG lipids, and 38.5% structured lipids. As yet another non-limiting example, lipid nanoparticles may comprise 55% cationic lipids, 10% non-cationic lipids, 2.5% PEG lipids, and 32.5% structured lipids. In some embodiments, the cationic lipid may be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0284] In some embodiments, the lipid nanoparticle formulations described herein may be four-component lipid nanoparticles. The lipid nanoparticles may comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticles may comprise 40-60% cationic lipid, 5-15% non-cationic lipid, 1-2% PEG lipid, and 30-50% structured lipid. As another non-limiting example, the lipid nanoparticles may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid, and 38.5% structured lipid. As yet another non-limiting example, the lipid nanoparticles may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid, and 32.5% structured lipid. In some embodiments, the cationic lipid may be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0285] In some embodiments, the lipid nanoparticle formulations described herein may contain a cationic lipid, a non-cationic lipid, a PEG lipid, and a structural lipid. As a non-limiting example, the lipid nanoparticles may contain 50% cationic lipid DLin-KC2-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structural lipid cholesterol. As a non-limiting example, the lipid nanoparticles may contain 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structural lipid cholesterol. As a non-limiting example, the lipid nanoparticles may contain 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DMG, and 38.5% structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparticles comprise 55% cationic lipid L319, 10% non-cationic lipid DSPC, 2.5% PEG lipid PEG-DMG, and 32.5% structural lipid cholesterol.

[0286] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or optional additional components in a vaccine composition can vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. For example, the composition may contain 0.1% to 99% (w / w) active ingredient. By way of example, the composition may contain 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, or at least 80% (w / w) active ingredient.

[0287] In some embodiments, an RNA vaccine composition can include a polynucleotide described herein and is formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC, and PEG2000-DMG, trisodium citrate buffer, sucrose, and water for injection. As a non-limiting example, the composition contains 2.0 mg / mL of a drug substance (e.g., a polynucleotide encoding VZV), 21.8 mg / mL of MC3, 10.1 mg / mL of cholesterol, 5.4 mg / mL of DSPC, 2.7 mg / mL of PEG2000-DMG, 5.16 mg / mL of trisodium citrate, 71 mg / mL of sucrose, and 1.0 mL of water for injection.

[0288] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10 to 500 nm, 20 to 400 nm, 30 to 300 nm, or 40 to 200 nm, or 50 to 150 nm, 50 to 200 nm, 80 to 100 nm, or 80 to 200 nm.

[0289] Liposomes, lipoplexes and lipid nanoparticles In some embodiments, the RNA vaccine pharmaceutical composition may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713), incorporated herein by reference in its entirety), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0290] In some embodiments, the RNA vaccine may be formulated in a lyophilized gel-phase liposome composition as described in U.S. Patent Application Publication No. US2012060293, which is incorporated herein by reference in its entirety.

[0291] Nanoparticle formulations can include phosphate conjugates.Phosphate conjugates can prolong the in vivo circulation time and / or enhance the targeted delivery of nanoparticles.The phosphate conjugates used in the present invention can be prepared by the method described in WO2013033438 or US Patent Application Publication No. US20130196948 (each of which is incorporated herein by reference in its entirety).By way of non-limiting example, the phosphate conjugate can include any one compound of the formula described in International Publication No. WO2013033438 (which is incorporated herein by reference in its entirety).

[0292] The nanoparticle formulation may include a polymer conjugate. The polymer conjugate may be a water-soluble conjugate. The polymer conjugate may have a structure described in U.S. Patent Application Publication No. 20130059360 (the entire contents of which are incorporated herein by reference). In some embodiments, the polymer conjugates of the polynucleotides of the present invention can be prepared using the methods and / or segmented polymer reagents described in U.S. Patent Application Publication No. 20130072709 (the entire contents of which are incorporated herein by reference). In other embodiments, the polymer conjugates may have pendant side groups comprising a ring moiety, such as, but not limited to, the polymer conjugates described in U.S. Patent Application Publication No. US20130196948 (the entire contents of which are incorporated herein by reference).

[0293] The nanoparticle formulation may include a conjugate that improves delivery of the nanoparticles of the present invention in a subject. Furthermore, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject. In some embodiments, the conjugate may be a "self" peptide designed from the human membrane protein CD47 (e.g., the "self" particle described by Rodriguez et al. (Science 2013, 339, 971-975, incorporated herein by reference in its entirety). As shown by Rodriguez et al., the self peptide delayed macrophage-mediated clearance of nanoparticles and improved nanoparticle delivery. In other embodiments, the conjugate may be the membrane protein CD47 (see, e.g., Rodriguez et al. Science 2013, 339, 971-975, incorporated herein by reference in its entirety). Rodriguez et al. showed that CD47, like the "self" peptide, can increase the rate of circulating particles in a subject compared to scrambled peptides and PEG-coated nanoparticles.

[0294] In some embodiments, the RNA vaccines of the present invention are formulated in nanoparticles containing a conjugate that improves delivery of the nanoparticles of the present invention in a subject. The conjugate may be a CD47 membrane, or the conjugate may be derived from a CD47 membrane protein, such as the "self" peptide described above. In other embodiments, the nanoparticles may contain PEG and a conjugate of CD47 or a derivative thereof. In yet other embodiments, the nanoparticles may contain both the "self" peptide described above and the membrane protein CD47.

[0295] In some embodiments, the "self" peptide and / or CD47 protein may be conjugated to a virus-like particle or pseudovirion, as described herein, for delivery of the RNA vaccines of the present invention.

[0296] In another embodiment, the RNA vaccine pharmaceutical composition comprises a polynucleotide of the present invention and a conjugate that may have a degradable bond. Non-limiting examples of conjugates include aromatic moieties containing ionizable hydrogen atoms, spacer moieties, and water-soluble polymers. Non-limiting examples of pharmaceutical compositions comprising conjugates with degradable bonds and methods for delivering the pharmaceutical compositions are described in U.S. Patent Application Publication No. US20130184443, the entire contents of which are incorporated herein by reference.

[0297] The nanoparticle formulation may be a carbohydrate nanoparticle comprising a carbohydrate carrier and an RNA vaccine. Non-limiting examples of carbohydrate carriers include, but are not limited to, anhydrous modified phytoglycogen or glycogen-type substances, phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydrous modified phytoglycogen β-dextrin (see, e.g., International Publication No. WO2012109121, the entire contents of which are incorporated herein by reference).

[0298] The nanoparticle formulations of the present invention may be coated with a surfactant or polymer to improve particle delivery. In some embodiments, the nanoparticles may be coated with a hydrophilic coating, such as, but not limited to, a PEG coating and / or a coating with a neutral surface charge. The hydrophilic coating may be useful for delivering nanoparticles containing large payloads, such as, but not limited to, RNA vaccines, into the central nervous system. As a non-limiting example, nanoparticles containing hydrophilic coatings and methods for making such nanoparticles are described in U.S. Patent Application Publication No. US20130183244, the entire contents of which are incorporated herein by reference.

[0299] In some embodiments, the lipid nanoparticles of the present invention can be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods for making hydrophilic polymer particles are described in U.S. Patent Application Publication No. US20130210991, the entire contents of which are incorporated herein by reference.

[0300] In other embodiments, the lipid nanoparticles of the present invention may be hydrophobic polymer particles.

[0301] Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids known as rapidly cleared lipid nanoparticles (reLNPs). Ionic cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and can be potential toxicogens. Rapid metabolism of rapidly cleared lipids can improve the tolerability and therapeutic index of lipid nanoparticles in rats by a dose of 1 mg / kg to 10 mg / kg. The inclusion of enzymatically degradable ester bonds can improve the degradation and metabolic profile of the cationic component while maintaining the activity of reLNP formulations. The ester bond can be internal to the lipid chain or can be located terminally at the end of the lipid chain. Internal ester bonds can replace any carbon in the lipid chain.

[0302] In some embodiments, the internal ester bond may be located on either side of the saturated carbon.

[0303] In some embodiments, an immune response can be elicited by delivering lipid nanoparticles that can include nanospecies, polymers, and immunogens (U.S. Patent Application Publication No. 20120189700 and International Publication No. WO2012099805, each of which is incorporated by reference in its entirety).

[0304] The polymer may encapsulate or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, modified RNA, and / or a polynucleotide as described herein. In some embodiments, the lipid nanoparticles may be formulated for use in vaccines, including, but not limited to, vaccines against pathogens.

[0305] Lipid nanoparticles may be engineered to modify their surface properties to allow them to cross mucosal barriers. Mucus is located in mucosal tissues, including, but not limited to, the oral cavity (e.g., the membranes of the cheeks and esophagus and tonsils), eyes, gastrointestinal tract (e.g., the stomach, small intestine, large intestine, colon, and rectum), nose, respiratory tract (e.g., membranes of the nose, pharynx, trachea, and bronchi), and reproductive tract (e.g., membranes of the vagina, cervix, and urethra). Nanoparticles larger than 10-200 nm are desirable for their high drug encapsulation efficiency and ability to provide sustained delivery of a wide range of drugs, but are considered too large to rapidly diffuse across mucosal barriers. Because mucus is continuously secreted, excreted, discarded, digested, and recycled, the majority of trapped particles can be removed from mucosal tissue within seconds or even hours. Large polymer nanoparticles (200-500 nm in diameter) densely coated with low-molecular-weight polyethylene glycol (PEG) diffused through mucus only 4-6 times slower than the same particles in water (Lai et al. PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171, each of which is incorporated herein by reference in its entirety). Nanoparticle transport can be determined using translocation velocity and / or fluorescence microscopy, including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high-resolution multiple particle tracking (MPT). As a non-limiting example, compositions capable of crossing mucosal barriers can be prepared as described in U.S. Pat. No. 8,241,670 or International Publication No. WO2013110028, each of which is incorporated herein by reference in its entirety.

[0306] Lipid nanoparticles engineered to pass through mucus may comprise polymeric materials (e.g., polymer cores) and / or polymer-vitamin conjugates and / or triblock copolymers. Polymeric materials may include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric materials may be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Publication No. WO2013116804, the entire contents of which are incorporated herein by reference. The polymeric materials may also be irradiated. As a non-limiting example, the polymeric materials may be gamma-irradiated (see, e.g., International Publication No. WO201282165, the entire contents of which are incorporated herein by reference). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), and poly(D,L-lactide-co-PEO-co-D,L-lactide). poly(D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),Polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly (isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticles may be coated with or associated with copolymers such as, but not limited to, block copolymers (such as the branched polyether-polyamide block copolymers described in International Publication No. WO2013012476, which is incorporated herein by reference in its entirety) and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymers (see, e.g., U.S. Patent Application Publication Nos. 20120121718, 20100003337, and U.S. Patent No. 8,263,665, each of which is incorporated herein by reference in its entirety). The copolymer may be a GRAS (Generally Regarded as Safe) polymer, and the formation of the lipid nanoparticles may be achieved by:It can be produced in a way that does not generate new chemical entities.For example, lipid nanoparticles can include poloxamer-coated PLGA nanoparticles, which can still pass through human mucus quickly without forming new chemical entities (Yang et al. Angew. Chem. Int. Ed. 2011 50:25972600, the entire contents of which are incorporated herein by reference).A non-limiting and scalable method for producing nanoparticles that can pass through human mucus is described by Xu et al. (see, for example, J Control Release 2013,170(2):279-86, the entire contents of which are incorporated herein by reference).

[0307] The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin portion of the conjugate can be substituted with other suitable moieties, including, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, hydrophobic moieties, or hydrophobic components of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains, and alkylene oxide chains).

[0308] In some embodiments, the RNA (e.g., mRNA) vaccine pharmaceutical composition may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713, incorporated herein by reference in its entirety)), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0309] In some embodiments, the RNA vaccine may be formulated in a lyophilized gel-phase liposome composition as described in U.S. Patent Application Publication No. US2012060293, which is incorporated herein by reference in its entirety.

[0310] Nanoparticle formulations can include phosphate conjugates.Phosphate conjugates can extend the in vivo circulation time and / or increase the targeted delivery of nanoparticles.The phosphate conjugates used in the present invention can be prepared by the method described in WO2013033438 or US Patent Application Publication No. 20130196948 (each of which is incorporated herein by reference in its entirety).By way of non-limiting example, the phosphate conjugate can include any one compound of the formula described in International Publication No. WO2013033438 (which is incorporated herein by reference in its entirety).

[0311] The nanoparticle formulation may include a polymer conjugate. The polymer conjugate may be a water-soluble conjugate. The polymer conjugate may have a structure described in U.S. Patent Application Publication No. 20130059360 (the entire contents of which are incorporated herein by reference). In some embodiments, the polymer conjugates of the polynucleotides of the present invention can be prepared using the methods and / or segmented polymer reagents described in U.S. Patent Application Publication No. 20130072709 (the entire contents of which are incorporated herein by reference). In other embodiments, the polymer conjugates may have pendant side groups comprising a ring moiety, such as, but not limited to, the polymer conjugates described in U.S. Patent Application Publication No. US20130196948 (the entire contents of which are incorporated herein by reference).

[0312] The nanoparticle formulation may include a conjugate that improves delivery of the nanoparticles of the present invention in a subject. Furthermore, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject. In some embodiments, the conjugate may be a "self" peptide designed from the human membrane protein CD47 (e.g., the "self" particle described by Rodriguez et al. (Science 2013, 339, 971-975), incorporated herein by reference in its entirety). As shown by Rodriguez et al., the self peptide delayed macrophage-mediated clearance of nanoparticles and improved nanoparticle delivery. In other embodiments, the conjugate may be the membrane protein CD47 (see, e.g., Rodriguez et al. Science 2013, 339, 971-975, incorporated herein by reference in its entirety). Rodriguez et al. showed that CD47, like the "self" peptide, can increase the proportion of circulating particles in a subject compared to scrambled peptides and PEG-coated nanoparticles.

[0313] In some embodiments, the RNA vaccines of the present invention are formulated in nanoparticles containing a conjugate that improves delivery of the nanoparticles of the present disclosure in a subject. The conjugate may be a CD47 membrane, or the conjugate may be derived from a CD47 membrane protein, such as the "self" peptide described above. In other embodiments, the nanoparticles may contain PEG and a conjugate of CD47 or a derivative thereof. In yet other embodiments, the nanoparticles may contain both the "self" peptide described above and the membrane protein CD47.

[0314] In some embodiments, the "self" peptide and / or CD47 protein may be conjugated to a virus-like particle or pseudovirion, as described herein, for delivery of the RNA vaccines of the present invention.

[0315] In another embodiment, the RNA vaccine pharmaceutical composition comprises a polynucleotide of the present invention and a conjugate that may have a degradable bond. Non-limiting examples of conjugates include aromatic moieties containing ionizable hydrogen atoms, spacer moieties, and water-soluble polymers. Non-limiting examples of pharmaceutical compositions comprising conjugates with degradable bonds and methods for delivering the pharmaceutical compositions are described in U.S. Patent Application Publication No. US20130184443, the entire contents of which are incorporated herein by reference.

[0316] The nanoparticle formulation may be a carbohydrate nanoparticle comprising a carbohydrate carrier and an RNA (e.g., mRNA) vaccine. Non-limiting examples of carbohydrate carriers include, but are not limited to, anhydrous modified phytoglycogen or glycogen-type substances, phytoglycogen octenyl succinate, phytoglycogen β-dextrin, or anhydrous modified phytoglycogen β-dextrin (see, e.g., International Publication No. WO2012109121, the entire contents of which are incorporated herein by reference).

[0317] The nanoparticle formulations of the present invention may be coated with a surfactant or polymer to improve particle delivery. In some embodiments, the nanoparticles may be coated with a hydrophilic coating, such as, but not limited to, a PEG coating and / or a coating with a neutral surface charge. The hydrophilic coating may be useful for delivering nanoparticles containing large payloads, such as, but not limited to, RNA vaccines, into the central nervous system. As a non-limiting example, nanoparticles containing hydrophilic coatings and methods for making such nanoparticles are described in U.S. Patent Application Publication No. US20130183244, the entire contents of which are incorporated herein by reference.

[0318] In some embodiments, the lipid nanoparticles of the present invention can be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods for making hydrophilic polymer particles are described in U.S. Patent Application Publication No. US20130210991, the entire contents of which are incorporated herein by reference.

[0319] In other embodiments, the lipid nanoparticles of the present invention may be hydrophobic polymer particles.

[0320] Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids known as rapidly cleared lipid nanoparticles (reLNPs). Ionic cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and can be potential toxicogens. Rapid metabolism of rapidly cleared lipids can improve the tolerability and therapeutic index of lipid nanoparticles in rats by a dose of 1 mg / kg to 10 mg / kg. The inclusion of enzymatically degradable ester bonds can improve the degradation and metabolic profile of the cationic component while maintaining the activity of reLNP formulations. The ester bond can be internal to the lipid chain or can be located terminally at the end of the lipid chain. Internal ester bonds can replace any carbon in the lipid chain.

[0321] In some embodiments, the internal ester bond may be located on either side of the saturated carbon.

[0322] In some embodiments, an immune response can be elicited by delivering lipid nanoparticles that can include nanospecies, polymers, and immunogens (U.S. Patent Application Publication No. 20120189700 and International Publication No. WO2012099805, each of which is incorporated by reference in its entirety).

[0323] The polymer may encapsulate or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, modified RNA, and / or a polynucleotide as described herein. In some embodiments, the lipid nanoparticles may be formulated for use in vaccines, including, but not limited to, vaccines against pathogens.

[0324] Lipid nanoparticles may be engineered to modify their surface properties to allow them to cross mucosal barriers. Mucus is located in mucosal tissues, including, but not limited to, the oral cavity (e.g., the membranes of the cheeks and esophagus and tonsils), eyes, gastrointestinal tract (e.g., the stomach, small intestine, large intestine, colon, and rectum), nose, respiratory tract (e.g., membranes of the nose, pharynx, trachea, and bronchi), and reproductive tract (e.g., membranes of the vagina, cervix, and urethra). Nanoparticles larger than 10-200 nm are desirable for their high drug encapsulation efficiency and ability to provide sustained delivery of a wide range of drugs, but are considered too large to rapidly diffuse across mucosal barriers. Because mucus is continuously secreted, excreted, discarded, digested, and recycled, the majority of trapped particles can be removed from mucosal tissue within seconds or even hours. Large polymer nanoparticles (200-500 nm in diameter) densely coated with low-molecular-weight polyethylene glycol (PEG) diffused through mucus only 4-6 times slower than the same particles in water (Lai et al. PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171, each of which is incorporated herein by reference in its entirety). Nanoparticle transport can be determined using translocation velocity and / or fluorescence microscopy, including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high-resolution multiple particle tracking (MPT). As a non-limiting example, compositions capable of crossing mucosal barriers can be prepared as described in U.S. Pat. No. 8,241,670 or International Publication No. WO2013110028, each of which is incorporated herein by reference in its entirety.

[0325] Lipid nanoparticles engineered to pass through mucus may comprise a polymeric material (i.e., a polymer core) and / or a polymer-vitamin conjugate and / or a triblock copolymer. Polymeric materials may include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Publication No. WO2013116804, the entire contents of which are incorporated herein by reference. The polymeric material may also be irradiated. As a non-limiting example, the polymeric material may be gamma-irradiated (see, e.g., International Publication No. WO201282165, the entire contents of which are incorporated herein by reference). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), and poly(D,L-lactide-co-PEO-co-D,L-lactide). poly(D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),Polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly (isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticles may be coated with or associated with copolymers such as, but not limited to, block copolymers (such as the branched polyether-polyamide block copolymers described in International Publication No. WO2013012476, which is incorporated herein by reference in its entirety) and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymers (see, e.g., U.S. Patent Application Publication Nos. 20120121718 and 20100003337, and U.S. Patent No. 8,263,665, each of which is incorporated herein by reference in its entirety). The copolymer may be a GRAS (Generally Regarded as Safe) polymer, and the formation of the lipid nanoparticles may be achieved by:It can be produced in a way that does not generate new chemical entities.For example, lipid nanoparticles can include poloxamer-coated PLGA nanoparticles, which can still pass through human mucus quickly without forming new chemical entities (Yang et al. Angew. Chem. Int. Ed. 2011 50:25972600, the entire contents of which are incorporated herein by reference).A non-limiting and scalable method for producing nanoparticles that can pass through human mucus is described by Xu et al. (see, for example, J Control Release 2013,170(2):279-86, the entire contents of which are incorporated herein by reference).

[0326] The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin portion of the conjugate can be substituted with other suitable moieties, including, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, hydrophobic moieties, or hydrophobic components of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains, and alkylene oxide chains).

[0327] Lipid nanoparticles engineered to penetrate mucus can include surface modifiers such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4 dornase alfa, neltenexin, erdosteine), and various DNases, including rhDNase. The surface modifier may be embedded in the particle surface, enmeshed in the particle surface, or disposed on the surface of the lipid nanoparticle (e.g., by coating, adsorption, covalent bonding, or other process) (see, e.g., U.S. Patent Application Publication Nos. 20100215580 and 20080166414 and US20130164343, the contents of each of which are incorporated by reference in their entirety).

[0328] In some embodiments, the mucus-penetrating lipid nanoparticles may contain at least one polynucleotide described herein. The polynucleotide may be encapsulated in the lipid nanoparticle and / or disposed on the surface of the particle. The polynucleotide may be covalently bound to the lipid nanoparticle. The mucus-penetrating lipid nanoparticle formulation may contain multiple nanoparticles. Furthermore, the formulation may contain particles that can interact with mucus and alter the structural properties and / or adhesiveness of the surrounding mucus to reduce mucoadhesion, which may increase delivery of the mucus-penetrating lipid nanoparticles to mucosal tissue.

[0329] In other embodiments, the mucus-penetrating lipid nanoparticles can be hypotonic formulations that include a coating that enhances mucosal passage. The formulation can be hypotonic relative to the epithelium to which it is delivered.

[0330] Non-limiting examples of hypotonic formulations can be found in International Publication No. WO2013110028, the entire contents of which are incorporated herein by reference.

[0331] In some embodiments, to improve delivery across mucosal barriers, the RNA vaccine formulation may include or be a hypotonic solution, which has been shown to increase the rate at which mucus-inert particles, including but not limited to mucus-penetrating particles, can reach the vaginal epithelial surface (see, e.g., Ensign et al. Biomaterials 2013, 34(28):6922-9, the entire contents of which are incorporated herein by reference).

[0332] In some embodiments, the RNA vaccine is formulated as a lipoplex, including, but not limited to, the ATUPLEX™ system, DACC system, DBTC system, and other siRNA lipoplex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, MA), and targeted and non-targeted nucleic acid delivery based on polyethyleneimine (PEI) or protamine (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol.Ther.2010 23:334-344;Kaufmann et al.Microvasc Res 2010 80:286-293;Weide et al.J Immunother.2009 32:498-507;Weide et al.J Immunother.2008 31:180-188;Pascolo,Expert Opin.Biol.Ther.4:1285-1294;Fotin-Mleczek et al.,2011 J.Immunother.34:1-15;Song et al.,Nature Biotechnol.2005,23:709-717;Peer et al.,Proc Natl Acad Sci US A.2007 6;104:4095-4100;deFougerolles Hum Gene Ther.2008 19:125-132, each of which is incorporated herein by reference in its entirety).

[0333] In some embodiments, such formulations can also be constructed or engineered to passively or actively target various cell types in vivo, including, but not limited to, hepatocytes, immune cells, tumor cells, endothelial cells, antigen-presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, each of which is incorporated by reference in its entirety. An example of passive targeting of formulations to hepatocytes includes DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA based lipid nanoparticle formulations, which have been shown to bind to apolipoprotein E and promote hepatocyte binding and uptake of the formulation in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364, incorporated herein by reference in its entirety).Formulations can also be selectively targeted by expressing different ligands on their surface, as exemplified by, but not limited to, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeting approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319;Akinc et al.,Mol Ther.2010 18:1357-1364;Srinivasan et al.,Methods Mol Biol.2012 820:105-116;Ben-Arie et al.,Methods Mol Biol.2012 757:497-507;Peer 2010 J Control Release.20:63-68;Peer et al.,Proc Natl Acad Sci US A.2007 104:4095-4100;Kim et al.,Methods Mol Biol.2011 721:339-353;Subramanya et al.,Mol Ther.2010 18:2028-2037;Song et al.,Nat Biotechnol.2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, each of which is incorporated herein by reference in its entirety).

[0334] In some embodiments, RNA (e.g., mRNA) vaccines are formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLNs) can be spherical with an average diameter of up to 1000 nm. SLNs have a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. In other embodiments, lipid nanoparticles can be self-assembled lipid-polymer nanoparticles (see Zhang et al., ACS Nano, 2008, 2(8), pp. 1696-1702, the entire contents of which are incorporated herein by reference). As a non-limiting example, SLNs can be those described in International Publication No. WO2013105101 (the entire contents of which are incorporated herein by reference). As another non-limiting example, SLNs can be prepared by the methods or processes described in International Publication No. WO2013105101 (the entire contents of which are incorporated herein by reference).

[0335] Although this formulation can increase cell transfection and / or increase the translation of encoded proteins by RNA vaccines, liposomes, lipoplexes, or lipid nanoparticles can be used to improve the efficiency of polynucleotide-targeted protein production. One such example is the use of lipid encapsulation to enable effective systemic delivery of polyplexed plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720, the entire contents of which are incorporated herein by reference). Liposomes, lipoplexes, or lipid nanoparticles can also be used to increase the stability of polynucleotides.

[0336] In some embodiments, the RNA (e.g., mRNA) vaccines of the present invention can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a release profile of a pharmaceutical composition or compound that conforms to a specific release pattern that achieves a therapeutic outcome. In some embodiments, the RNA vaccines can be encapsulated in a delivery material described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulate" means to entrap, surround, or encase. When referring to formulations of the compounds of the present invention, encapsulation can be substantial, complete, or partial. The term "substantially encapsulated" means that at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, greater than 99.9, or greater than 99.999% of the pharmaceutical composition or compound of the present invention is encapsulated, surrounded, or encased within the delivery material. "Partial encapsulation" means that less than 10%, 10, 20, 30, 40, 50, or less of the pharmaceutical composition or compound of the present invention is trapped, surrounded, or enveloped within the delivery material. Advantageously, encapsulation can be determined by measuring leakage or activity of the pharmaceutical composition or compound of the present invention using fluorescence and / or electron microscopy. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or more than 99.99% of the pharmaceutical composition or compound of the present invention is encapsulated within the delivery material.

[0337] In some embodiments, the controlled release formulation may include, but is not limited to, a triblock copolymer. As a non-limiting example, the formulation may include two different types of triblock copolymers (International Publication Nos. WO2012131104 and WO2012131106, the entire contents of each of which are incorporated herein by reference).

[0338] In other embodiments, the RNA vaccine can be encapsulated in a lipid nanoparticle or rapidly cleared lipid nanoparticle, which can then be encapsulated within a polymer, hydrogel, and / or surgical sealant described herein and / or known in the art. By way of non-limiting example, the polymer, hydrogel, or surgical sealant can be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc., Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego, CA), fibrinogen polymer (Ethicon Inc., Cornelia, GA), TISSELL® (Baxter International, Inc., Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc., Deerfield, IL).

[0339] In other embodiments, the lipid nanoparticles may be encapsulated within any polymer known in the art that can form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticles may be encapsulated within a polymer matrix that may be biodegradable.

[0340] In some embodiments, RNA vaccine formulations for controlled release and / or targeted delivery may also include at least one controlled-release coating, including, but not limited to, OPADRY®, polyvinylpyrrolidone / vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS®, and cellulose derivatives such as aqueous ethylcellulose dispersions (AQUACOAT® and SURELEASE®).

[0341] In some embodiments, the RNA (e.g., mRNA) vaccine controlled release and / or targeted delivery formulation may include at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In other embodiments, the degradable polyester may include a PEG conjugate to form a PEGylated polymer.

[0342] In some embodiments, an RNA vaccine controlled release and / or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and / or PEG-related polymer derivative as described in U.S. Pat. No. 8,404,222, the entire contents of which are incorporated herein by reference.

[0343] In other embodiments, the RNA vaccine controlled release delivery formulation comprising at least one polynucleotide may be a controlled release polymer system as described in U.S. Patent Application Publication No. 20130130348, which is incorporated herein by reference in its entirety.

[0344] In some embodiments, the RNA (e.g., mRNA) vaccines of the present invention can be encapsulated in therapeutic nanoparticles, referred to herein as "therapeutic nanoparticle RNA vaccines." Therapeutic nanoparticles can be prepared using methods described herein and known in the art, including, but not limited to, International Publication Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, U.S. Patent Application Publication Nos. US20110262491, US20100104645, US20100087337, US20100087337, and US20100087337. Nos. 20100068285, 20110274759, 20100068286, 20120288541, 20130123351, and 20130230567, as well as U.S. Patent Nos. 8,206,747, 8,293,276, 8,318,208, and 8,318,211, the contents of each of which are incorporated herein by reference in their entirety. In other embodiments, therapeutic polymeric nanoparticles can be identified by the methods described in U.S. Patent Application Publication No. US20120140790, the contents of which are incorporated herein by reference in their entirety.

[0345] In some embodiments, therapeutic nanoparticle RNA vaccines can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that follows a release rate over a specific period of time. The specific period of time includes, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained-release nanoparticles can include a polymer and a therapeutic agent, such as, but not limited to, a polynucleotide of the present invention (see International Publication No. 2010075072 and U.S. Patent Application Publication Nos. US20100216804, US20110217377, and US20120201859, each of which is incorporated herein by reference in its entirety). In another non-limiting example, a sustained-release formulation can include an agent that allows sustained bioavailability, such as, but not limited to, a crystal, a macromolecular gel, and / or a microparticle suspension (see U.S. Patent Application Publication No. US20130150295, the entire contents of which are incorporated herein by reference).

[0346] In some embodiments, therapeutic nanoparticle RNA vaccines can be formulated to be target-specific. As a non-limiting example, the therapeutic nanoparticles can include corticosteroids (see International Publication No. WO2011084518, which is incorporated herein by reference in its entirety). As a non-limiting example, the therapeutic nanoparticles can be formulated into nanoparticles described in International Publication Nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521, and U.S. Patent Application Publication Nos. US20100069426, US20120004293, and US20100104655 (each of which is incorporated herein by reference in its entirety).

[0347] In some embodiments, the nanoparticles of the present invention may comprise a polymer matrix. By way of non-limiting example, the nanoparticles may comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or a combination thereof.

[0348] In some embodiments, the therapeutic nanoparticles may comprise a diblock copolymer. In some embodiments, the diblock copolymer may comprise PEG in combination with a polymer such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or a combination thereof. In yet other embodiments, the diblock copolymer may be a high-X diblock copolymer, such as those described in International Publication No. WO2013120052, the entire contents of which are incorporated herein by reference.

[0349] As a non-limiting example, the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Patent Application Publication No. US20120004293 and U.S. Patent No. 8,236,330, each of which is incorporated herein by reference in its entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968 and International Publication No. WO2012166923, each of which is incorporated herein by reference in its entirety). In yet another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in U.S. Patent Application Publication No. 20130172406, the entire contents of which are incorporated herein by reference.

[0350] In some embodiments, therapeutic nanoparticles can include multiblock copolymers (see, e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Application Publication No. 20130195987, the contents of each of which are incorporated by reference in their entirety).

[0351] In yet another non-limiting example, the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see, e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) used as a delivery vehicle for the TGF-β1 gene in Lee et al., "Thermosensitive Hydrogel as a TGF-β1 Gene Delivery Vehicle Enhances Diabetic Wound Healing," Pharmaceutical Research, 2003 20(12):1995-2000, and as a controlled gene delivery system in Li et al., "Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel," Pharmaceutical Research, 2003 20(6):884-888; and Chang et al., "Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle," J. Controlled Release. 2007 118:245-253, each of which is incorporated herein by reference in its entirety). The RNA (e.g., mRNA) vaccines of the present disclosure can be formulated in lipid nanoparticles comprising PEG-PLGA-PEG block copolymers.

[0352] In some embodiments, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer (see, e.g., U.S. Patent Application Publication No. 20120076836, which is incorporated herein by reference in its entirety).

[0353] In some embodiments, the therapeutic nanoparticles may comprise at least one acrylic polymer, including, but not limited to, acrylic acid, methacrylic acid, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof.

[0354] In some embodiments, the therapeutic nanoparticles may comprise at least one poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be a copolymer, such as a random copolymer. As a non-limiting example, the random copolymer may have a structure such as those described in International Publication No. WO2013032829 or U.S. Patent Application Publication No. 20130121954 (the entire contents of which are incorporated herein by reference). In some embodiments, the poly(vinyl ester) polymer may be conjugated to a polynucleotide described herein. In other embodiments, the poly(vinyl ester) polymer that can be used in the present invention may be one described herein.

[0355] In some embodiments, the therapeutic nanoparticles may comprise at least one diblock copolymer. The diblock copolymer may be, but is not limited to, a poly(lactic) acid-poly(ethylene) glycol copolymer (see, e.g., International Publication No. WO2013044219, incorporated herein by reference in its entirety). As a non-limiting example, the therapeutic nanoparticles can be used to treat cancer (see, e.g., International Publication No. WO2013044219, incorporated herein by reference in its entirety).

[0356] In some embodiments, the therapeutic nanoparticles can include at least one cationic polymer described herein and / or known in the art.

[0357] In some embodiments, therapeutic nanoparticles may comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(amidoamine) dendrimers, poly(β-aminoesters) (see, e.g., U.S. Pat. No. 8,287,849, incorporated herein by reference in its entirety), and combinations thereof. In other embodiments, the nanoparticles described herein may comprise amine-cationic lipids, such as those described in International Publication No. WO2013059496, incorporated herein by reference in its entirety. In some embodiments, the cationic lipids may have an amino-amine or amino-amide moiety.

[0358] In some embodiments, the therapeutic nanoparticles may comprise at least one degradable polyester, which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In other embodiments, the degradable polyester may comprise a PEG conjugate to form a PEGylated polymer.

[0359] In other embodiments, the therapeutic nanoparticles may comprise a conjugate of at least one targeting ligand, which may be any ligand known in the art, such as, but not limited to, a monoclonal antibody (Kirpotin et al., Cancer Res. 2006 66:6732-6740, incorporated herein by reference in its entirety).

[0360] In some embodiments, therapeutic nanoparticles may be formulated in an aqueous solution and used to target cancer (see International Publication No. WO2011084513 and U.S. Patent Application Publication No. 20110294717, each of which is incorporated by reference in its entirety).

[0361] In some embodiments, therapeutic nanoparticle RNA vaccines (e.g., therapeutic nanoparticles comprising at least one RNA vaccine) can be formulated using the methods described by Podobinski et al. in U.S. Pat. No. 8,404,799, the entire contents of which are incorporated herein by reference.

[0362] In some embodiments, RNA (e.g., mRNA) vaccines may be encapsulated in, bound to, and / or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Publication Nos. WO2010005740, WO2012149454, and WO2013019669, and U.S. Patent Application Publication Nos. US20110262491, US20100104645, US20100087337, and US20120244222 (each of which is incorporated by reference in its entirety). Synthetic nanocarriers can be formulated using methods known in the art and / or described herein. By way of non-limiting example, synthetic nanocarriers can be formulated by the methods described in International Publication Nos. WO2010005740, WO2010030763, and WO201213501, and U.S. Patent Application Publication Nos. US20110262491, US20100104645, US20100087337, and US2012024422, each of which is incorporated by reference in its entirety. In other embodiments, synthetic nanocarrier formulations can be lyophilized using the methods described in International Publication No. WO2011072218 and U.S. Patent No. 8,211,473, the contents of each of which are incorporated by reference in their entirety. In still other embodiments, formulations of the present invention, including but not limited to synthetic nanocarriers, may be lyophilized or reconstituted by the methods described in U.S. Patent Application Publication No. 20130230568, the entire contents of which are incorporated herein by reference.

[0363] In some embodiments, synthetic nanocarriers may contain reactive groups that release the polynucleotides described herein (see International Publication No. WO20120952552 and U.S. Patent Application Publication No. US20120171229, each of which is incorporated by reference in its entirety).

[0364] In some embodiments, synthetic nanocarriers may contain immunostimulatory agents that enhance the immune response from delivery of the synthetic nanocarriers. As a non-limiting example, synthetic nanocarriers may include Th1 immunostimulatory agents that may enhance the Th1-based response of the immune system (see International Publication No. WO2010123569 and U.S. Patent Application Publication No. 20110223201, each of which is incorporated by reference in its entirety).

[0365] In some embodiments, synthetic nanocarriers can be formulated for targeted release. In some embodiments, synthetic nanocarriers are formulated to release polynucleotides at a specified pH and / or after a desired time interval. As a non-limiting example, synthetic nanoparticles can be formulated to release RNA vaccines after 24 hours and / or at pH 4.5 (International Publication Nos. WO2010138193 and WO2010138194 and U.S. Patent Application Publication Nos. US20110020388 and US20110027217, each of which is incorporated by reference in its entirety).

[0366] In some embodiments, synthetic nanocarriers can be formulated for controlled and / or sustained release of the polynucleotides described herein. By way of non-limiting example, synthetic nanocarriers for sustained release can be formulated by methods known in the art, methods described herein, and / or methods described in International Publication No. WO2010138192 and U.S. Patent Application Publication No. 20100303850 (each of which is incorporated by reference in its entirety).

[0367] In some embodiments, the RNA vaccines may be formulated for controlled and / or sustained release, and the formulation comprises at least one polymer that is a crystallizable side chain (CYSC) polymer. CYSC polymers are described in U.S. Patent No. 8,399,007, which is incorporated herein by reference in its entirety.

[0368] In some embodiments, synthetic nanocarriers can be formulated for use as vaccines. In some embodiments, synthetic nanocarriers can encapsulate at least one polynucleotide encoding at least one antigen. As a non-limiting example, a synthetic nanocarrier can include at least one antigen and an excipient for a vaccine formulation (see International Publication No. WO2011150264 and US Patent Application Publication No. 20110293723, each of which is incorporated by reference in its entirety). As another non-limiting example, a vaccine formulation can include at least two synthetic nanocarriers containing the same or different antigens and an excipient (see International Publication No. WO2011150249 and US Patent Application Publication No. 20110293701, each of which is incorporated by reference in its entirety). Vaccine dosage forms may be selected by methods described herein, methods known in the art, and / or methods described in International Publication No. WO2011150258 and U.S. Patent Application Publication No. US20120027806, each of which is incorporated by reference in its entirety.

[0369] In some embodiments, synthetic nanocarriers may contain at least one polynucleotide encoding at least one adjuvant. As a non-limiting example, the adjuvant may include dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, dimethyldioctadecylammonium phosphate, or dimethyldioctadecylammonium acetate (DDA), and the non-polar fraction of a mycobacterial total lipid extract or a portion of the non-polar fraction (see, e.g., U.S. Patent No. 8,241,610, incorporated herein by reference in its entirety). In other embodiments, synthetic nanocarriers may contain at least one polynucleotide and an adjuvant. As a non-limiting example, synthetic nanocarriers containing adjuvants may be formulated by the methods described in International Publication No. WO2011150240 and U.S. Patent Application Publication No. US20110293700 (each of which is incorporated herein by reference in its entirety).

[0370] In some embodiments, synthetic nanocarriers may encapsulate at least one polynucleotide encoding a peptide, fragment, or region derived from a virus. Non-limiting examples of synthetic nanocarriers include those described in International Publication Nos. WO2012024621, WO201202629, WO2012024632, and U.S. Patent Application Publication Nos. US20120064110, US20120058153, and US20120058154 (each of which is incorporated by reference in its entirety).

[0371] In some embodiments, synthetic nanocarriers may be conjugated to polynucleotides that may be capable of eliciting a humoral response and / or a cytotoxic T lymphocyte (CTL) response (see, e.g., International Publication No. WO2013019669, incorporated herein by reference in its entirety).

[0372] In some embodiments, the RNA vaccine may be encapsulated in, bound to, and / or associated with a zwitterionic lipid. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Application Publication No. 20130216607, the entire contents of which are incorporated herein by reference. In some embodiments, zwitterionic lipids can be used in the liposomes and lipid nanoparticles described herein.

[0373] In some embodiments, the RNA vaccines may be formulated in colloidal nanocarriers, as described in US Patent Application Publication No. 20130197100, the entire contents of which are incorporated herein by reference.

[0374] In some embodiments, the nanoparticles can be optimized for oral administration. The nanoparticles can include at least one cationic biopolymer, such as, but not limited to, chitosan or its derivatives. As a non-limiting example, the nanoparticles can be formulated by the method described in U.S. Patent Application Publication No. 20120282343 (incorporated herein by reference in its entirety).

[0375] In some embodiments, LNPs comprise the lipid KL52 (an amino lipid disclosed in U.S. Patent Application Publication No. 2012 / 0295832, expressly incorporated herein by reference in its entirety). The activity and / or safety of LNP administration (as determined by examining one or more of ALT / AST, white blood cell count, and cytokine induction) may be improved by incorporating such a lipid. KL52-containing LNPs may be administered intravenously and / or in one or more doses. In some embodiments, administration of KL52-containing LNPs results in the same or improved mRNA and / or protein expression compared to LNPs containing MC3.

[0376] In some embodiments, RNA vaccines may be delivered using even smaller LNPs, such as particles with diameters of less than 0.1 μm up to 100 nm, including but not limited to, less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, and less than 250 μm. , less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm or less than 975 μm.

[0377] In other embodiments, the RNA (e.g., mRNA) vaccine is about 1 nm to about 100 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm. , about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 4 0nm, about 5nm to about 50nm, about 5nm to about 60nm, about 5nm to about 70nm, about 5nm to about 80nm, about 5nm to about 90nm, about 10 to about 5 20 to about 80 nm, about 30 to about 80 nm, about 40 to about 90 nm, about 50 to about 90 nm, about 60 to about 90 nm, about 60 to about 90 nm, about 70 to about 90 nm, about 20 to about 50 nm, about 30 to about 50 nm, about 40 to about 50 nm, about 20 to about 60 nm, about 30 to about 60 nm, about 40 to about 60 nm, about 20 to about 70 nm, about 30 to about 70 nm, about 40 to about 70 nm, about 50 to about 70 nm, about 60 to about 70 nm, about 20 to about 80 nm, about 30 to about 80 nm, about 40 to about 80 nm, about 50 to about 80 nm, about 60 to about 80 nm, about 20 to about 90 nm, about 30 to about 90 nm, about 40 to about 90 nm, about 50 to about 90 nm, about 60 to about 90 nm, and / or about 70 to about 90 nm.

[0378] In some embodiments, such LNPs are synthesized using methods that include a microfluidic mixer. Exemplary microfluidic mixers include, but are not limited to, slit-type interdigital micromixers, such as those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or zigzag herringbone micromixers (SHM). (Zhigaltsev, IV et al., "Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing" has been published. (Langmuir. 2012.28:3633-40; Belliveau, NM et al., "Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA." Molecular Therapy-Nucleic Acids. 2012.1:e37; Chen, D. et al., "Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation." J Am Chem. Soc. 2012.134(16):6948-51, each of which is incorporated herein by reference in its entirety).

[0379] In some embodiments, the method for producing LNPs with SHM further includes mixing at least two input streams, where the mixing occurs via microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels that exist in a herringbone pattern, causing rotational flow and overlapping of the fluids. This method can also include a surface for fluid mixing, where the surface changes direction while the fluid circulates. Methods for producing LNPs using SHM include those disclosed in U.S. Patent Application Publication Nos. 2004 / 0262223 and 2012 / 0276209 (each of which is expressly incorporated by reference in its entirety).

[0380] In some embodiments, the RNA vaccines of the present invention may be formulated into lipid nanoparticles generated using a micromixer such as, but not limited to, a slit-type interdigital microstructure mixer (SIMM-V2) or a standard slit-type interdigital micromixer (SSIMM) or a caterpillar (CPMM) or a jet impingement (IJMM) (Institut fur Mikrotechnik Mainz GmbH, Mainz, Germany).

[0381] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure can be formulated in lipid nanoparticles generated using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442:368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647-651, each of which is incorporated herein by reference in its entirety). As a non-limiting example, controlled microfluidic formulation involves passive mixing of low Reynolds number, steady-pressure driven flows within microchannels (see, e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647-651, each of which is incorporated herein by reference in its entirety).

[0382] In some embodiments, the RNA (e.g., mRNA) vaccines of the present invention can be formulated into lipid nanoparticles generated using a micromixer chip, such as those manufactured by Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). Micromixer chips can be used to rapidly mix two or more fluid streams through a slit and recombination mechanism.

[0383] In some embodiments, the RNA (e.g., mRNA) vaccines of the present invention can be formulated for delivery using drug-encapsulated microspheres as described in International Publication No. WO2013063468 or U.S. Patent No. 8,440,614 (each of which is incorporated herein by reference in its entirety). The microspheres can contain compounds of formula (I), (II), (III), (IV), (V), or (VI) as described in International Publication No. WO2013063468 (the entire contents of which are incorporated herein by reference). In other embodiments, amino acids, peptides, polypeptides, and lipids (APPLs) are useful for delivering the RNA vaccines of the present invention to cells (see International Publication No. WO2013063468, the entire contents of which are incorporated herein by reference).

[0384] In some embodiments, the RNA (e.g., mRNA) vaccine of the present disclosure has a diameter of about 10 to about 100 nm, for example, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 20 to about 90 nm They may be formulated into lipid nanoparticles having a diameter of about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm.

[0385] In some embodiments, the lipid nanoparticles may have a diameter of about 10 to 500 nm.

[0386] In some embodiments, the lipid nanoparticles may have a diameter of greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, 950 nm, or greater than 1000 nm.

[0387] In some embodiments, the lipid nanoparticles may be critical size lipid nanoparticles described in International Publication No. WO2013059922 (the entire contents of which are incorporated herein by reference). Critical size lipid nanoparticles may comprise a lipid bilayer surrounding an aqueous or hydrophobic core, where the lipid bilayer may comprise phospholipids such as, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In other embodiments, critical size lipid nanoparticles may comprise polyethylene glycol lipids such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0388] In some embodiments, the RNA vaccine may be delivered, localized, and / or concentrated at a specific location using the delivery methods described in International Publication No. WO2013063530 (the entire contents of which are incorporated herein by reference). As a non-limiting example, empty polymer particles may be administered to a subject before, simultaneously with, or after delivery of the RNA vaccine to the subject. Upon contact with the subject, the empty polymer particles change volume and become lodged, embedded, immobilized, or trapped at a specific location within the subject.

[0389] In some embodiments, the RNA vaccine may be formulated in an active agent release system (see, e.g., U.S. Patent Application Publication No. US20130102545, the entire contents of which are incorporated herein by reference). The active agent release system may include 1) at least one nanoparticle bound to an oligonucleotide inhibitor strand that hybridizes with a catalytically active nucleic acid, and 2) a compound bound to at least one substrate molecule that is bound to a therapeutically active substance (e.g., a polynucleotide described herein), where the therapeutically active substance is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0390] In some embodiments, RNA (e.g., mRNA) vaccines can be formulated in nanoparticles comprising an inner core comprising non-cellular material and an outer surface comprising a cell membrane. The cell membrane can be derived from a cell or a virus. As a non-limiting example, nanoparticles can be produced by the method described in International Publication No. WO2013052167 (incorporated herein by reference in its entirety). As another non-limiting example, the nanoparticles described in International Publication No. WO2013052167 (incorporated herein by reference in its entirety) can be used to deliver the RNA vaccines described herein.

[0391] In some embodiments, the RNA vaccine can be formulated in a lipid bilayer (original cell) supported on a porous nanoparticle. Original cells are described in International Publication No. WO2013056132, the entire contents of which are incorporated herein by reference.

[0392] In some embodiments, the RNA vaccines described herein can be formulated in polymeric nanoparticles described in U.S. Patent Nos. 8,420,123 and 8,518,963 and European Patent No. EP2073848B1 (the entire contents of each of which are incorporated herein by reference), or in polymeric nanoparticles made by the methods described therein. As a non-limiting example, polymeric nanoparticles such as those described in U.S. Patent No. 8,518,963 (the entire contents of which are incorporated herein by reference), or made by the methods describe...

Claims

1. A Varicella-Zoster Virus (VZV) vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one VZV antigenic polypeptide, and a pharma- ceutically acceptable carrier or excipient.

2. 2. The VZV vaccine of claim 1, wherein the at least one RNA polynucleotide has an open reading frame encoding two or more VZV antigenic polypeptides.

3. 3. The VZV vaccine of claim 1 or 2, wherein the at least one VZV antigenic polypeptide is a VZV glycoprotein or an antigenic fragment or epitope thereof.

4. 3. The VZV vaccine of claim 1 or 2, wherein the at least one VZV antigenic polypeptide is a VZV tegument protein or an antigenic fragment or epitope thereof.

5. 4. The VZV vaccine of claim 3, wherein the VZV glycoprotein is selected from VZV gE, gI, gB, gH, gK, gL, gC, gN and gM.

6. The VZV vaccine of claim 5 , wherein the VZV glycoprotein is VZV gE.

7. The VZV vaccine of claim 5 , wherein the VZV glycoprotein is VZV gI.

8. 7. The VZV vaccine of claim 6, wherein the VZV glycoprotein is a variant VZV gE polypeptide.

9. 9. The VZV vaccine of claim 8, wherein the variant VZV gE polypeptide is a truncated polypeptide lacking the anchor domain (ER retention domain).

10. 9. The VZV vaccine of claim 8, wherein the variant VZV gE polypeptide is a truncated polypeptide lacking the carboxy-terminal tail domain.

11. 10. The VZV vaccine of claim 9, wherein the VZV antigenic fragment is a truncated VZV gE polypeptide comprising amino acids 1-561 of SEQ ID NO:

10.

12. 11. The VZV vaccine of claim 10, wherein the VZV antigenic fragment is a VZV gE variant polypeptide comprising amino acids 1-573 of SEQ ID NO:

18.

13. 9. The VZV vaccine of claim 8, wherein the variant VZV gE polypeptide has at least one mutation in at least one motif associated with ER retention, endocytosis and / or localization to the Golgi apparatus or trans-Golgi network.

14. 14. The VZV vaccine of claim 13, wherein the variant VZV gE polypeptide has at least one mutation in at least one phosphorylated acidic motif.

15. 14. The VZV vaccine of claim 13, wherein the variant VZV gE polypeptide has a Y582G mutation relative to SEQ ID NO:

10.

16. 14. The VZV vaccine of claim 13, wherein the variant VZV gE polypeptide has a Y569A mutation relative to SEQ ID NO:

10.

17. 14. The VZV vaccine of claim 13, wherein the variant VZV gE polypeptide has a Y582G mutation and a Y569A mutation relative to SEQ ID NO:

10.

18. The VZV vaccine of claim 12, wherein the VZV antigenic fragment has a Y569A mutation relative to SEQ ID NO:

10.

19. 18. The VZV vaccine of claim 6 or any one of claims 13 to 17, wherein the variant VZV gE polypeptide has the sequence A-E-A-A-D-A (SEQ ID NO:58).

20. The VZV vaccine of claim 6 or any one of claims 13 to 17, wherein the variant VZV gE polypeptide has an Igκ sequence at the C-terminus.

21. The VZV vaccine of claim 11 or 12, wherein the VZV gE antigenic fragment has an Igκ sequence at the C-terminus.

22. The VZV vaccine of claim 5 , wherein said at least one RNA polynucleotide encodes antigenic polypeptides of VZV gE and gI.

23. The VZV vaccine of claim 6 or any one of claims 8 to 21, comprising at least one RNA polynucleotide having an open reading frame encoding a gI antigenic polypeptide.

24. 22. The VZV vaccine of claim 6 or any one of claims 8 to 21, comprising at least one RNA polynucleotide having an open reading frame encoding an antigenic polypeptide selected from VZV gB, gH, gK, gL, gC, gN and gM.

25. 5. The VZV vaccine of claim 4, wherein at least one RNA polynucleotide has an open reading frame encoding a VZV antigenic polypeptide selected from VZV gE, gI, gB, gH, gK, gL, gC, gN and gM.

26. The VZV vaccine of any one of claims 5 to 25, further comprising a live attenuated VZV, a whole inactivated VZV or a VZV VLP.

27. The VZV vaccine of any one of claims 1 to 3, wherein the at least one RNA polynucleotide is encoded by at least one nucleic acid sequence, at least one fragment of a nucleic acid sequence, or at least one epitope of a nucleic acid sequence selected from SEQ ID NOs: 1-8, 37, 39, 41, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90, or the at least one RNA polynucleotide is encoded by at least one nucleic acid sequence, at least one fragment of a nucleic acid sequence, or at least one epitope of a nucleic acid sequence selected from SEQ ID NOs: 6, 37, 39, 84, or 86.

28. The VZV vaccine of any one of claims 1 to 3, wherein the at least one RNA polynucleotide comprises a sequence selected from SEQ ID NOs: 92-108 and 123-131.

29. The VZV vaccine of any one of claims 1 to 3, wherein the at least one RNA polynucleotide comprises at least one fragment of a nucleic acid sequence or at least one epitope of an mRNA sequence selected from SEQ ID NOs: 92-108 and 123-131.

30. The VZV vaccine of claim 6 , wherein the at least one RNA polynucleotide is encoded by SEQ ID NO:

1.

31. The VZV vaccine of claim 7 , wherein the at least one RNA polynucleotide is encoded by SEQ ID NO:

2.

32. The VZV vaccine of claim 11 , wherein the at least one RNA polynucleotide is encoded by SEQ ID NO:

3.

33. The VZV vaccine of claim 12, wherein the at least one RNA polynucleotide is encoded by SEQ ID NO:

5.

34. 20. The VZV vaccine of claim 18, wherein the at least one RNA polynucleotide is encoded by SEQ ID NO: 6, 37, 39, 84 or 86.

35. 20. The VZV vaccine of claim 19, wherein the at least one RNA polynucleotide is encoded by SEQ ID NO:

7.

36. 20. The VZV vaccine of claim 19, wherein the RNA polynucleotide is encoded by SEQ ID NO:

8.

37. The VZV vaccine of claim 3, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having at least 90% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 26, 30, 42 and 45-55.

38. 4. The VZV vaccine of claim 3, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having at least 95% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 26, 30, 38, 42 and 45-55.

39. 4. The VZV vaccine of claim 3, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having at least 96% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 26, 30, 38, 42 and 45-55.

40. 4. The VZV vaccine of claim 3, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having at least 97% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 26, 30, 38, 42 and 45-55.

41. 4. The VZV vaccine of claim 3, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having at least 98% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 26, 30, 38, 42 and 45-55.

42. 4. The VZV vaccine of claim 3, wherein the at least one RNA polynucleotide encodes an antigenic polypeptide having at least 99% identity to the amino acid sequence of any of SEQ ID NOs: 10, 14, 26, 30, 38, 42 and 45-55.

43. The VZV vaccine of any one of claims 1 to 42, wherein the open reading frame by which the polynucleotide is encoded is codon optimized.

44. The VZV vaccine of any one of claims 1 to 43, which is multivalent.

45. The VZV vaccine of any one of claims 1 to 43, wherein the RNA polynucleotide comprises at least one chemical modification.

46. 46. ​​The VZV vaccine of claim 45, further comprising a second chemical modification.

47. 47. The VZV vaccine of claim 45 or 46, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

48. 46. ​​The VZV vaccine of claim 45, wherein 80% of the uracils in the open reading frame have a chemical modification.

49. 49. The VZV vaccine of claim 48, wherein 100% of the uracils in the open reading frame have a chemical modification.

50. The VZV vaccine of any one of claims 45 to 49, wherein the chemical modification is at the 5-position of the uracil.

51. 51. The VZV vaccine of claim 50, wherein the chemical modification is N1-methylpseudouridine.

52. 52. The VZV vaccine of any one of claims 1 to 51, formulated in cationic lipid nanoparticles.

53. 53. The VZV vaccine of claim 52, wherein the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

54. 54. The VZV vaccine of claim 53, wherein said cationic lipid is an ionizable cationic lipid, said non-cationic lipid is a neutral lipid, and said sterol is cholesterol.

55. 53. The VZV vaccine of claim 52, wherein the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

56. 55. The VZV vaccine of claim 54, wherein the cationic lipid nanoparticles have a molar ratio of about 20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid.

57. 53. The VZV vaccine of claim 52, wherein the nanoparticles have a polydispersity value of less than 0.

4.

58. 53. The VZV vaccine of claim 52, wherein the nanoparticles have a neutral net charge at neutral pH.

59. 53. The VZV vaccine of claim 52, wherein the nanoparticles have an average diameter of 50-200 nm.

60. A VZV vaccine described in any one of claims 1 to 59 for use in a method of inducing an antigen-specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen-specific immune response.

61. Use of a VZV vaccine described in any one of claims 1 to 59 in the manufacture of a medicament for use in a method for inducing an antigen-specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen-specific immune response.

62. A method for preventing or treating a VZV infection comprising administering to a subject a vaccine according to any one of claims 1 to 59.

63. 60. A method of inducing an antigen-specific immune response in a subject, comprising administering to the subject a vaccine according to any one of claims 1 to 59 in an amount effective to produce an antigen-specific immune response in the subject.

64. 64. The method of claim 63, wherein the antigen-specific immune response comprises a T cell response or a B cell response.

65. 65. The method of claim 63 or 64, wherein the subject receives a single dose of the vaccine.

66. 65. The method of claim 63 or 64, wherein the subject receives an initial dose and a second (booster) dose of the vaccine.

67. 67. The method of any one of claims 63 to 66, wherein the vaccine is administered to the subject by intradermal or intramuscular injection.

68. The method of any one of claims 63 to 67, wherein the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by at least 1 log compared to a control.

69. The method of any one of claims 63 to 68, wherein the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by 1 to 3 logs compared to a control.

70. The method of any one of claims 63 to 69, wherein the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by at least 2-fold compared to a control.

71. The method of any one of claims 63 to 70, wherein the titer of anti-antigenic polypeptide antibodies produced in the subject is increased by at least 2-10 fold compared to a control.

72. The method of any one of claims 68 to 71, wherein the control is the titer of anti-antigenic polypeptide antibodies produced in a subject who has not been vaccinated against the virus.

73. 72. The method of any one of claims 68 to 71, wherein the control is the titer of anti-antigenic polypeptide antibodies produced in a subject who has been administered a live attenuated or inactivated vaccine against the virus.

74. 72. The method of any one of claims 68 to 71, wherein the control is the titer of anti-antigenic polypeptide antibodies produced in a subject who has been administered a recombinant or purified protein vaccine against the virus.

75. The method of any one of claims 68 to 71, wherein the control is the titer of anti-antigenic polypeptide antibodies produced in a subject who has been administered a VLP vaccine against the virus.

76. The method of any one of claims 63 to 75, wherein the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant protein vaccine or a purified protein vaccine against the virus, reduced by at least half, and the titer of anti-antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a recombinant protein vaccine or a purified protein vaccine against the virus, respectively.

77. The method of any one of claims 63 to 75, wherein the effective amount is a dose equivalent to a standard therapeutic dose of a live attenuated or inactivated vaccine against the virus reduced by at least half, and the titer of anti-antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a live attenuated or inactivated vaccine against the virus, respectively.

78. The method of any one of claims 63 to 75, wherein the effective amount is a dose equivalent to a standard therapeutic dose of a VLP vaccine against the virus that is reduced by at least half, and the titer of anti-antigenic polypeptide antibodies produced in the subject is equivalent to the titer of anti-antigenic polypeptide antibodies produced in a control subject administered a standard therapeutic dose of a VLP vaccine against the virus.

79. 79. The method of any one of claims 63 to 78, wherein the effective amount is a total dose of 50 μg to 1000 μg.

80. 80. The method of claim 79, wherein the effective amount is a dose of 25 μg, 100 μg, 400 μg or 500 μg administered to the subject a total of two times.

81. 81. The method of any one of claims 63 to 80, wherein the efficacy of the vaccine against the virus is greater than 65%.

82. 82. The method of any one of claims 63-81, wherein the vaccine immunizes the subject against the virus for up to two years.

83. 82. The method of any one of claims 63-81, wherein the vaccine immunizes the subject against the virus for more than two years.

84. 84. The method of any one of claims 63 to 83, wherein the subject is aged between 1 year and about 10 years.

85. 85. The method of any one of claims 63-84, wherein the subject has been exposed to the virus, the subject is infected with the virus, or the subject is at risk of infection by the virus.

86. The method of any one of claims 63 to 85, wherein the subject is immunocompromised.

87. 60. An engineered nucleic acid encoding at least one RNA polynucleotide of the vaccine of any one of claims 1 to 59.

88. A VZV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having (i) a 5' terminal cap, (ii) an open reading frame encoding at least one VZV antigenic polypeptide, and (iii) a 3' poly A tail.

89. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide is encoded by a sequence comprising the sequence identified by SEQ ID NO:

6.

90. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide is encoded by a sequence comprising the sequence identified by SEQ ID NO:

37.

91. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide is encoded by a sequence comprising the sequence identified by SEQ ID NO:

39.

92. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide is encoded by a sequence comprising the sequence identified by SEQ ID NO:

84.

93. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide is encoded by a sequence comprising the sequence identified by SEQ ID NO:

86.

94. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide comprises a sequence identified by SEQ ID NO:

128.

95. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide comprises a sequence identified by SEQ ID NO: 99 or 133.

96. 89. The vaccine of claim 88, wherein the at least one mRNA polynucleotide comprises a sequence identified by SEQ ID NO: 107 or 134.

97. 89. The vaccine of claim 88, wherein the at least one VZV antigenic polypeptide comprises the sequence identified by SEQ ID NO:

38.

98. 98. The vaccine of any one of claims 88 to 97, wherein the 5' end cap is or comprises 7mG(5')ppp(5')N1mpNp.

99. 99. The vaccine of any one of claims 88-98, wherein 100% of the uracils in the open reading frame are modified to contain an N1-methylpseudouridine at position 5 of the uracil.

100. 100. The vaccine of any one of claims 88 to 99, formulated in lipid nanoparticles.

101. 101. The vaccine of claim 100, further comprising trisodium citrate buffer, sucrose and water.

102. 1. A VZV vaccine comprising at least one messenger ribonucleic acid (mRNA) polynucleotide comprising a nucleic acid sequence identified by SEQ ID NO:99, having (i) a 5' end cap, (ii) an open reading frame encoding a VZV antigenic polypeptide, and (iii) a 3' polyA tail, wherein the 5' end cap is 7mG(5')ppp(5')NlmpNp.

103. 103. The VZV vaccine of claim 102, wherein the uracil nucleotide of the sequence identified by SEQ ID NO:99 is modified to contain an N1-methylpseudouridine at position 5 of the uracil nucleotide.

104. 1. A VZV vaccine comprising at least one messenger ribonucleic acid (mRNA) polynucleotide comprising a nucleic acid sequence identified by SEQ ID NO: 133, having (i) a 5' end cap, (ii) an open reading frame encoding a VZV antigenic polypeptide, and (iii) a 3' polyA tail, wherein the 5' end cap is 7mG(5')ppp(5')NlmpNp.

105. 105. The VZV vaccine of claim 104, wherein the uracil nucleotide of the sequence identified by SEQ ID NO: 133 is modified to contain an N1-methylpseudouridine at position 5 of the uracil nucleotide.

106. 1. A VZV vaccine comprising at least one messenger ribonucleic acid (mRNA) polynucleotide comprising a nucleic acid sequence identified by SEQ ID NO: 107, having (i) a 5' end cap, (ii) an open reading frame encoding a VZV antigenic polypeptide, and (iii) a 3' polyA tail, wherein the 5' end cap is 7mG(5')ppp(5')NlmpNp.

107. 107. The VZV vaccine of claim 106, wherein the uracil nucleotide of the sequence identified by SEQ ID NO: 107 is modified to contain an N1-methylpseudouridine at position 5 of the uracil nucleotide.

108. 1. A VZV vaccine comprising at least one messenger ribonucleic acid (mRNA) polynucleotide comprising a nucleic acid sequence identified by SEQ ID NO:134, having (i) a 5' end cap, (ii) an open reading frame encoding a VZV antigenic polypeptide, and (iii) a 3' polyA tail, wherein the 5' end cap is 7mG(5')ppp(5')NlmpNp.

109. 109. The VZV vaccine of claim 108, wherein the uracil nucleotide of the sequence identified by SEQ ID NO: 134 is modified to contain an N1-methylpseudouridine at position 5 of the uracil nucleotide.

110. 1. A pharmaceutical composition for use in vaccinating a subject, comprising: containing an effective amount of mRNA encoding a Varicella-Zoster Virus (VZV) antigen; The effective amount is sufficient to result in a detectable level of antigen when measured in the subject's serum 1 to 72 hours after administration of the pharmaceutical composition.

111. The composition of claim 110, wherein the antigen has a cutoff index of 1 to 2.

112. 1. A pharmaceutical composition for use in vaccinating a subject, comprising: containing an effective amount of mRNA encoding a Varicella-Zoster Virus (VZV) antigen; The pharmaceutical composition, wherein the effective amount is sufficient to result in a neutralizing titer of 1,000 to 10,000 caused by neutralizing antibodies against the antigen when measured in the serum of the subject 1 to 72 hours after administration.

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