Human cytomegalovirus vaccine

Abstract

To provide HCMV ribonucleic acid (RNA) vaccines, as well as methods for using the vaccines and compositions comprising the vaccines.SOLUTION: The HCMV RNA vaccines of the present disclosure may be used to induce a balanced immune response against human cytomegalovirus comprising both cellular and humoral immunity, without many of the risks associated with DNA or attenuated virus vaccination.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 245,166, filed October 22, 2015, entitled "Human Cytomegalovirus Vaccine," and U.S. Provisional Application No. 62 / 247,614, filed October 28, 2015, entitled "Human Cytomegalovirus Vaccine," and U.S. Provisional Application No. 62 / 245,031, filed October 22, 2015, the disclosures of each of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Human cytomegalovirus (HCMV) is a genus of viruses in the order Herpesvirales, family Herpesviridae, and subfamily Betaherpesvirinae. This genus currently contains eight species that have been identified and classified in different mammals, including humans, monkeys, and rodents. The most studied genus is human cytomegalovirus, also known as human herpesvirus 5 (HHV-5), which is widely distributed in the human population. HHV-5-associated diseases include mononucleosis and pneumonia. All herpesviruses share the characteristic ability to maintain internal latency for extended periods of time. While they may be found throughout the body, CMV infections are often associated with the salivary glands of humans and other mammals. Other CMV viruses are found in several mammalian species, but species isolated from animals differ from HCMV in terms of genome structure and have not been reported to cause human disease.

[0003] HCMV is endemic in most parts of the world. It is a ubiquitous, large, enveloped virus that infects 50–100% of the adult population worldwide. While generally asymptomatic in immunocompromised hosts, HCMV infection is a major cause of morbidity and mortality in infants and subsequently in immunocompromised individuals, such as those with congenital or neonatal infection, transplant recipients, or AIDS patients.

[0004] Primary infection usually causes asymptomatic disease, after which the virus becomes latent and retains the ability to reactivate later. The virus is transmitted through bodily fluids such as blood, saliva, urine, semen, and breast milk. Presymptomatic or immunocompromised individuals are particularly susceptible to HCMV infection. It is estimated that at least 60% of the US population has been exposed to CMV, with a greater than 90% incidence rate in high-risk groups (e.g., fetuses infected by CMV during pregnancy or HIV-infected individuals).

[0005] In healthy individuals, HCMV typically causes asymptomatic infection or mild flu-like symptoms. However, in two populations, HCMV can cause significant pathology. First, HCMV is a leading cause of congenital defects in newborns infected in utero. Of congenitally infected newborns, 5–10% have major clinical manifestations at birth, including microcephaly, intracranial calcifications, hepatitis, and cytomegalic inclusion disease, which affects many tissues and organs, including the central nervous system, liver, and retina, and can lead to multiple organ failure and death. Other infants may be asymptomatic at birth but later develop hearing loss or central nervous system abnormalities, particularly intellectual disability and mental retardation. These pathologies are due in part to the ability of HCMV to invade and replicate in diverse cell types, including epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, neurons, and monocytes / macrophages.

[0006] A second population at risk is immunocompromised patients, such as those with HIV infection and those undergoing transplants. In this setting, the virus becomes an opportunistic pathogen, causing severe disease with high morbidity and mortality. Clinical disease results in a variety of symptoms, including fever, pneumonia, hepatitis, encephalitis, myelitis, colitis, uveitis, retinitis, and neuropathy. Rare manifestations of HCMV infection in immunocompromised individuals include Guillain-Barré syndrome, meningoencephalitis, pericarditis, myocarditis, thrombocytopenia, and hemolytic anemia. Furthermore, HCMV infection increases the risk of organ graft loss due to transplant vascular sclerosis and restenosis and may increase atherosclerosis in transplant patients and the general population. HCMV infection is estimated to cause clinical disease in 75% of patients in the first year after transplant.

[0007] Currently, there is no approved HCMV vaccine. Two candidate vaccines, Towne and gB / MF59, have completed phase II efficacy trials. The Towne vaccine is protective against both infection and disease caused by challenge with the virulent Toledo strain and also appears to be effective in preventing severe posttransplant CMV disease. However, in a small phase II clinical trial, a low-dose Towne vaccine failed to protect against infection in children whose mothers were actively shed CMV but were seronegative.

[0008] The gB / MF59 vaccine is a protein subunit vaccine composed of a membrane-defective version of the HCMV gB protein that induces high levels of fibroblast entry-neutralizing antibodies in humans and has been shown to be safe and well-tolerated in both adults and young children. A recent phase II, double-blind, placebo-controlled trial of the gB / MF59 vaccine demonstrated 50% efficacy in inducing sterilizing immunity. Because the vaccine induces strong antibody responses but very weak T-cell responses, the partial efficacy conferred by the vaccine is thought to be primarily antibody-mediated. While this HCMV vaccine is the first to demonstrate protective efficacy, its 50% protection falls short of the 80–90% desired level for most vaccines.

[0009] Furthermore, antibody therapy has been used to control HCMV infection in immunocompromised individuals and reduce the pathological consequences of maternal-fetal infection, but such treatment is usually insufficient to eradicate the virus. HCMV immunoglobulin (Ig) has been administered to transplant patients in conjunction with immunosuppressive therapy for the prevention of HCMV disease with mixed results. Antibody therapy has also been used to control congenital infection and prevent neonatal disease. However, these products are plasma derivatives with relatively low efficacy and must be administered by intravenous infusion at very high doses to deliver sufficient amounts of neutralizing antibodies.

[0010] HCMV is a leading viral cause of neurodevelopmental abnormalities and other birth defects in children, resulting in substantial costs to society. While antiviral therapies are available, antiviral treatment is imperfect, and development of a CMV vaccine is the most promising strategy for preventing CMV infection. Given the significant health and economic benefits of an effective HCMV vaccine, the Institute of Medicine and the National Vaccine Program Office have classified CMV vaccine development as a top priority, although no candidate vaccines have been considered for licensing. Summary of the Invention [Problem to be solved by the invention]

[0011] summary In light of the lack of an HCMV vaccine, there is a significant need for a vaccine that is safe and effective in all patient populations to prevent and / or treat HCMV infection. In particular, a safe and effective vaccine is needed for immunocompromised women at risk of pregnancy and infant patients to prevent or reduce the severity and / or duration of HCMV. Provided herein is an RNA vaccine that builds on the knowledge that ribonucleic acid (e.g., messenger RNA (mRNA)) can safely guide the body's cellular machinery to produce almost any protein of interest, from natural proteins to antibodies and other entirely novel protein constructs that may have therapeutic activity both inside and outside the cell. The HCMV RNA vaccine of the present disclosure can be used to induce a balanced immune response against human cytomegalovirus, including both cellular and humoral immunity, without the many risks associated with DNA or attenuated virus vaccination. [Means for solving the problem]

[0012] RNA vaccines can be used in a variety of situations, depending on the prevalence of infection or the degree or level of unmet medical need. RNA vaccines can be used to treat and / or prevent various genotypes, strains, and isolates of HCMV. RNA vaccines have superior properties in that they generate significantly greater antibody titers and produce faster responses than commercially available antiviral therapeutic treatments. Without wishing to be bound by theory, RNA vaccines as mRNA polynucleotides are thought to be better designed to generate appropriate protein conformations during translation, since RNA vaccines share natural cellular machinery. Unlike traditional vaccines that are produced ex vivo and can induce undesirable cellular responses, RNA vaccines are presented to cell systems in a more natural manner.

[0013] Various human cytomegalovirus amino acid sequences encompassed by this disclosure are set forth below in Tables 1, 2, and 6. The RNA vaccines provided herein can comprise at least one RNA polynucleotide encoding at least one of the HCMV proteins provided in Tables 1, 2, or 6, or a fragment, homolog (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% identity) or derivative thereof.

[0014] Some embodiments of the present disclosure provide HCMV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide HCMV vaccines comprising at least one RNA polynucleotide having an open reading frame encoding two or more HCMV antigen polypeptides or immunogenic fragments or epitopes thereof. Some embodiments of the present disclosure provide HCMV vaccines comprising two or more RNA polynucleotides having open reading frames encoding two or more HCMV antigen polypeptides or immunogenic fragments or epitopes thereof. The one or more HCMV antigen polypeptides may be encoded on a single RNA polynucleotide or may be individually encoded by multiple (e.g., two or more) RNA polynucleotides.

[0015] In some embodiments, the antigenic polypeptide is an HCMV glycoprotein. For example, the HCMV glycoprotein can be selected from HCMV gH, gL, gB, gO, gN, and gM, and immunogenic fragments or epitopes thereof. In some embodiments, the antigenic polypeptide is an HCMV gH polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gL polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gB polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gO polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gN polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gM polypeptide. In some embodiments, the HCMV glycoprotein is encoded by the nucleic acid sequence of 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:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, or SEQ ID NO:113.

[0016] In some embodiments, the HCMV glycoprotein is a mutant gH polypeptide, a mutant gL polypeptide, or a mutant gB polypeptide. In some embodiments, the mutant HCMV gH, gL, or gB polypeptide is a truncated polypeptide lacking one or more of the following domain sequences: (1) a hydrophobic membrane proximal domain, (2) a transmembrane domain, and (3) a cytoplasmic domain. In some embodiments, the truncated HCMV gH, gL, or gB polypeptide lacks the hydrophobic membrane proximal domain, the transmembrane domain, and the cytoplasmic domain. In some embodiments, the truncated HCMV gH, gL, or gB polypeptide comprises only the extracellular domain sequence. In some embodiments, the HCMV truncated glycoprotein is encoded by the nucleic acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0017] In some embodiments, the antigenic polypeptide is an HCMV protein selected from UL83, UL123, UL128, UL130, and UL131A, or an immunogenic fragment or epitope thereof. In some embodiments, the antigenic polypeptide is an HCMV UL83 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL123 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL128 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL130 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL131A polypeptide. In some embodiments, the HCMV protein is encoded by the nucleic acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0018] In some embodiments, the antigenic polypeptide comprises two or more HCMV proteins, fragments thereof, or epitopes. In some embodiments, the antigenic polypeptide comprises two or more glycoproteins, fragments thereof, or epitopes. In some embodiments, the antigenic polypeptide comprises at least one HCMV glycoprotein, fragment thereof, or epitope and at least one other HCMV protein, fragment thereof, or epitope. In some embodiments, the two or more HCMV polypeptides are encoded by a single RNA polynucleotide. In some embodiments, the two or more HCMV polypeptides are encoded by two or more RNA polynucleotides, e.g., each HCMV polypeptide is encoded by a separate RNA polynucleotide. In some embodiments, the two or more HCMV glycoproteins may be any combination of HCMV gH, gL, gB, gO, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gB and one or more HCMV polypeptides selected from gH, gL, gO, gN, and gM polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gH and one or more HCMV polypeptides selected from gL, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gL and one or more HCMV polypeptides selected from gB, gH, gO, gN, and gM polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV glycoproteins are gB and gH. In some embodiments, the two or more HCMV glycoproteins are gB and gL. In some embodiments, the two or more HCMV glycoproteins are gH and gL. In some embodiments, the two or more HCMV glycoproteins are gB, gL, and gH.In some embodiments, the two or more HCMV proteins may be any combination of HCMV UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV glycoproteins are UL123 and UL130. In some embodiments, the two or more HCMV glycoproteins are UL123 and 131A. In some embodiments, the two or more HCMV glycoproteins are UL130 and 131A. In some embodiments, the two or more HCMV glycoproteins are UL128, UL130, and 131A. In some embodiments, the two or more HCMV proteins may be any combination of HCMV gB, gH, gL, gO, gM, gN, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gH and one or more HCMV polypeptides selected from gL, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more glycoproteins may be any combination of HCMV gL and one or more HCMV polypeptides selected from gH, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV glycoproteins are gL, gH, UL128, UL130, and 131A. In any of these embodiments in which the vaccine comprises two or more HCMV proteins, the HCMV gH may be a mutant gH, e.g., any mutant HCMV gH glycoprotein disclosed herein, e.g., any mutant HCMV disclosed in the preceding paragraph and in the Examples. In any of these embodiments in which the vaccine comprises two or more HCMV proteins, the HCMV gB may be a mutant gB, e.g., any mutant HCMV gB glycoprotein disclosed herein, e.g., any mutant HCMV gB disclosed in the preceding paragraph and in the Examples.In any of these embodiments in which the vaccine comprises two or more HCMV gL proteins, the HCMV gL may be a mutant gL, e.g., any mutant HCMV gL glycoprotein disclosed herein, e.g., any mutant HCMV gL disclosed in the previous paragraph and examples.

[0019] In certain embodiments, the HCMV vaccine comprises two or more RNA polynucleotides having open reading frames encoding two or more HCMV 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 protein encoded by a separate RNA polynucleotide), the two or more HCMV proteins are HCMV proteins selected from mutant gB, e.g., any of the mutant gB polypeptides disclosed herein in the preceding paragraph, and gH, gL, gO, gM, gN, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV proteins are HCMV proteins selected from mutant gH, e.g., any of the mutant gH polypeptides disclosed herein in the preceding paragraph, and gH, gL, gO, gM, gN, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV proteins are mutant gH, e.g., any of the mutant gH polypeptides disclosed herein in the previous paragraph, and an HCMV protein selected from gH, gL, gO, gM, gN, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the mutant HCMV proteins are mutant HCMV gB, mutant HCMV gL, and mutant HCMV gH, the mutant HCMV polypeptide is a truncated polypeptide selected from the following truncated polypeptides: lacking the hydrophobic membrane proximal domain; lacking the transmembrane domain; lacking the cytoplasmic domain; lacking two or more of the hydrophobic membrane proximal, transmembrane, and cytoplasmic domains; and including only the extracellular domain.

[0020] In some embodiments, an HCMV vaccine comprises a multimeric RNA polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or an immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or an immunogenic fragment or epitope thereof, wherein the 5'UTR of the RNA polynucleotide comprises a patterned UTR. In some embodiments, the patterned UTR has a repeating or alternating pattern such as ABABAB, or AABBAABBAABB, or ABCABCABC, or variants thereof repeated one, two, or three or more times. In these patterns, each letter A, B, or C represents a UTR that differs at the nucleotide level. In some embodiments, the 5'UTR of an RNA polynucleotide (e.g., a first nucleic acid) has a region of complementarity with a UTR of another RNA polynucleotide (a second nucleic acid). For example, the UTR nucleotide sequences of two polynucleotides to be linked (e.g., in a multimeric molecule) may be modified to contain regions of complementarity so that the two UTRs hybridize to form a multimeric molecule.

[0021] In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV antigen polypeptide is modified to allow for the formation of a multimeric sequence. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from UL128, UL130, and UL131A1 is modified to allow for the formation of a multimeric sequence. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV glycoprotein is modified to allow for the formation of a multimeric sequence. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV glycoprotein selected from gH, gL, gB, gO, gM, and gN is modified to allow for the formation of a multimeric sequence. In any of these embodiments, the multimer may be a dimer, trimer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Thus, in some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131A1 is modified to allow for dimer formation. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131A1 is modified to allow for trimer formation. In some embodiments, the 5'UTR of an RNA polynucleotide encoding an HCMV protein selected from gH, gL, gB, gO, gM, gN, UL128, UL130, and UL131A1 is modified to allow pentamer formation. Exemplary HCMV nucleic acids having modified 5'UTR sequences for the formation of multimeric molecules (e.g., dimers, trimers, pentamers, etc.) include SEQ ID NOs: 19-26.

[0022] In any of the above embodiments, the HCMV RNA polynucleotide may further comprise additional sequences, for example, one or more linker sequences or one or more sequence tags, such as a FLAG tag and a histidine tag.

[0023] Some embodiments of the present disclosure provide HCMV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having a single open reading frame encoding two or more (e.g., two, three, four, five, or more) HCMV antigen polypeptides or immunogenic fragments or epitopes thereof. Some embodiments of the present disclosure provide HCMV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having two or more open reading frames, e.g., two, three, four, five, or more open reading frames encoding two, three, four, five, or more HCMV antigen polypeptides. In any of these embodiments, the at least one RNA polynucleotide may encode two or more HCMV antigen polypeptides selected from gH, gB, gL, gO, gM, gN, UL83, UL123, UL128, UL130, UL131A, and fragments or epitopes thereof. In some embodiments, the at least one RNA polynucleotide encodes UL83 and UL123. In some embodiments, at least one RNA polynucleotide encodes gH and gL. In some embodiments, at least one RNA polynucleotide encodes UL128, UL130, and UL131A. In some embodiments, at least one RNA polynucleotide encodes gH, gL, UL128, UL130, and UL131A. In some embodiments, at least one RNA polynucleotide has a single open reading frame encoding two or more (e.g., 2, 3, 4, 5, or more) HCMV antigen polypeptides, the RNA polynucleotide further comprises an additional sequence, e.g., a linker sequence, or a sequence that aids in processing of the HCMV RNA transcript or polypeptide, e.g., a cleavage site sequence. In some embodiments, this additional sequence may be a protease sequence, such as a furin sequence. In some embodiments, the additional sequence may be a self-cleaving 2A peptide, such as a P2A, E2A, F2A, or T2A sequence.In some embodiments, the linker sequence and cleavage site sequence are interspersed between the sequences encoding the HCMV polypeptides. In some embodiments, the RNA polynucleotide is encoded by SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0024] In some embodiments, at least one RNA polynucleotide is encoded by at least one nucleic acid sequence selected from any of SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113, and homologs having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to a nucleic acid sequence selected from SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113. In some embodiments, at least one RNA polynucleotide is encoded by at least one nucleic acid sequence selected from any of SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113, and homologs having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity to a nucleic acid sequence selected from SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113. In some embodiments, the at least one RNA polynucleotide is encoded by at least a fragment of a nucleic acid sequence selected from any of SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113, and homologs having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to a nucleic acid sequence selected from SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, 68, and 108-113. In some embodiments, the at least one RNA polynucleotide is encoded by at least one nucleic acid sequence selected from any of the nucleic acid sequences disclosed herein, and homologs having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any of the nucleic acid sequences disclosed herein.

[0025] In any of the above embodiments in the previous paragraph, the HCMV RNA polynucleotide may further comprise additional sequences, for example, one or more linker sequences or one or more sequence tags, such as a FLAG tag and a histidine tag.

[0026] In some embodiments, 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: 32-52, 59, 61, 63, 65, 67, and 69. In some embodiments, 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: 32-52, 59, 61, 63, 65, 67, and 69. 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: 32-52, 59, 61, 63, 65, 67, and 69. In some embodiments, 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: 32-52, 59, 61, 63, 65, 67, and 69. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 98% identity to the amino acid sequence of SEQ ID NOs: 32-52, 59, 61, 63, 65, 67, and 69. In some embodiments, at least one RNA polynucleotide encodes an antigenic polypeptide having at least 99% identity to the amino acid sequence of SEQ ID NOs: 32-52, 59, 61, 63, 65, 67, and 69.

[0027] In some embodiments, the open reading frame encoding the HCMV polypeptide is codon-optimized. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein of SEQ ID NO: 32, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein of SEQ ID NO: 33, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein of SEQ ID NO: 34, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein of SEQ ID NO: 38, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein of SEQ ID NO: 40, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes an antigenic protein of SEQ ID NO: 42, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 47, and the RNA polynucleotide is a codon-optimized mRNA. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 50, and the RNA polynucleotide is a codon-optimized mRNA.

[0028] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 32, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 32, wherein the RNA polynucleotide has 80% or more identity to, but does not include, the wild-type mRNA sequence.

[0029] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 33, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 33, wherein the RNA polynucleotide has more than 80% identity to, but does not include, the wild-type mRNA sequence.

[0030] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 34, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 34, wherein the RNA polynucleotide has more than 80% identity to, but does not include, the wild-type mRNA sequence.

[0031] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 38, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 38, wherein the RNA polynucleotide has more than 80% identity to, but does not include, the wild-type mRNA sequence.

[0032] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 40, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 40, wherein the RNA polynucleotide has more than 80% identity to, but does not include, the wild-type mRNA sequence.

[0033] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 42, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence. In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 42, wherein the RNA polynucleotide has more than 80% identity to, but does not include, the wild-type mRNA sequence.

[0034] In some embodiments, at least one RNA polynucleotide is encoded by a sequence selected from SEQ ID NOs: 1-31 and comprises at least one chemical modification.

[0035] In some embodiments, the HCMV vaccine is multivalent. In some embodiments, the RNA polynucleotide comprises a polynucleotide sequence derived from a viral strain or isolate selected from VR1814 VR6952, VR3480B1 (ganciclovir resistant), VR4760 (ganciclovir and foscarnet resistant), Towne, TB40 / E, AD169, Merlin, and Toledo.

[0036] Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigen 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.

[0037] Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or immunogenic fragment thereof, wherein the at least one ribonucleic acid (RNA) polynucleotide has at least one chemical modification. In some embodiments, the at least one ribonucleic acid (RNA) polynucleotide further comprises a second chemical modification. In some embodiments, the at least one ribonucleic acid (RNA) polynucleotide having at least one chemical modification has a 5'-terminal 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.

[0038] Some embodiments of the present disclosure provide an HCMV vaccine comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigen 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, the chemical modification is N1-ethylpseudouridine.

[0039] Some embodiments of the present disclosure provide an HCMV vaccine formulated in a cationic lipid nanoparticle. In some embodiments, the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

[0040] In some embodiments, the cationic lipid is an ionizable 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).

[0041] In some embodiments, the lipid is [ka] is.

[0042] In some embodiments, the lipid is [ka] is.

[0043] 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 net neutral charge at neutral pH. In some embodiments, the nanoparticles have an average diameter of 50-200 nm.

[0044] Some embodiments of the present disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an amount of an HCMV RNA vaccine effective to generate 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 for generating an antigen-specific immune response comprises 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.

[0045] Also provided is an HCMV RNA 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 generate the antigen-specific immune response.

[0046] Further provided herein is the use of an HCMV RNA vaccine 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 to the subject an amount of the vaccine effective to generate an antigen-specific immune response.

[0047] Additionally provided is a method of preventing or treating HCMV infection comprising administering a vaccine of the present disclosure to a subject.

[0048] The HCMV vaccines disclosed herein can be formulated in an amount effective to generate an antigen-specific immune response in a subject.

[0049] In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least one log relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by one to three logs relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least two-fold compared to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least five-fold relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least ten-fold relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by two to ten-fold relative to a control.

[0050] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject who has not received the HCMV vaccine.

[0051] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject administered a live-attenuated or inactivated HCMV vaccine.

[0052] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject administered a recombinant or purified HCMV protein vaccine.

[0053] In some embodiments, the effective amount is a dose equivalent to at least a two-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0054] In some embodiments, the effective amount is a dose equivalent to at least a four-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0055] In some embodiments, the effective amount is a dose equivalent to at least a 10-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0056] In some embodiments, the effective amount is a dose equivalent to at least a 100-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0057] In some embodiments, the effective amount is a dose equivalent to at least a 1000-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0058] In some embodiments, the effective amount is a dose equivalent to a 2- to 1000-fold reduction of a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0059] In some embodiments, the effective amount is a total dose of 50 to 1000 μg. In some embodiments, the effective amount is a total dose of 100 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 100 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.

[0060] Another aspect of the present disclosure provides a method of inducing an antigen-specific immune response in a subject, comprising administering to the subject an HCMV vaccine disclosed herein in an amount effective to generate an antigen-specific immune response in the subject.

[0061] In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least one log relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by one to three logs relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least two-fold compared to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least five-fold relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least ten-fold relative to a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by two to ten-fold relative to a control.

[0062] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject who has not received the HCMV vaccine.

[0063] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject administered a live-attenuated or inactivated HCMV vaccine.

[0064] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject administered a recombinant or purified HCMV protein vaccine.

[0065] In some embodiments, the effective amount is a dose equivalent to at least a two-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant HCMV protein vaccine or a live attenuated HCMV vaccine.

[0066] In some embodiments, the effective amount is a dose equivalent to at least a four-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0067] In some embodiments, the effective amount is a dose equivalent to at least a 10-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0068] In some embodiments, the effective amount is a dose equivalent to at least a 100-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0069] In some embodiments, an effective amount is a dose equivalent to at least a 1000-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0070] In some embodiments, the effective amount is a dose equivalent to a 2- to 1000-fold reduction of a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0071] In some embodiments, the effective amount is a total dose of 50-1000 μg. In some embodiments, the effective amount is a total dose of 100 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 100 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.

[0072] Another aspect of the present disclosure provides an HCMV vaccine comprising a signal peptide linked to an HCMV antigenic polypeptide.

[0073] In some embodiments, the HCMV antigenic polypeptide is an HCMV glycoprotein or an antigenic fragment thereof. In some embodiments, the HCMV antigenic polypeptide is an HCMV gB, gM, gN, gH, gL, gO, UL83, UL123, UL128, UL130, or UL131A protein, or an antigenic fragment or epitope thereof. In some embodiments, the HCMV glycoprotein is selected from HCMV gB, gM, gN, gH, gL, and gO.

[0074] In some embodiments, the HCMV glycoprotein is HCMV gH. In some embodiments, the HCMV glycoprotein is HCMV gL. In some embodiments, the HCMV glycoprotein is HCMV gB. In some embodiments, the HCMV protein is HCMV UL128. In some embodiments, the HCMV protein is HCMV UL130. In some embodiments, the HCMV protein is HCMV UL131A. In some embodiments, the HCMV protein is HCMV UL83. In some embodiments, the HCMV protein is HCMV UL123. In some embodiments, the HCMV glycoprotein is a mutant HCMV gH polypeptide. In some embodiments, the HCMV glycoprotein is a mutant HCMV gL polypeptide. In some embodiments, the HCMV glycoprotein is a mutant HCMV gB polypeptide.

[0075] 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 amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 53).

[0076] In some embodiments, the signal peptide is an IgGκ signal peptide. In some embodiments, the signal peptide has the amino acid sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 54).

[0077] In some embodiments, the HCMV vaccine comprises at least one RNA polynucleotide encoding gH, gL, UL128, UL130, and UL131A, or an antigenic fragment or epitope thereof, and at least one RNA polynucleotide encoding gB, or an antigenic fragment or epitope thereof.

[0078] Further provided herein is the use of an HCMV vaccine to prevent congenital HCMV infection. Further provided herein is a method of administering an HCMV vaccine to a woman of childbearing age.

[0079] An embodiment of the present invention relates to a human cytomegalovirus (HCMV) vaccine comprising: i) at least one RNA polynucleotide having one or more open reading frames encoding HCMV antigenic polypeptides gH, gL, UL128, UL130 and / or UL131A, or antigenic fragments or epitopes thereof; ii) an RNA polynucleotide having an open reading frame encoding HCMV antigenic polypeptide gB, or an antigenic fragment or epitope thereof; and iii) a pharmaceutically acceptable carrier or excipient.

[0080] In some embodiments, the HCMV vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gH or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gL, or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL128, or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL130, or an antigenic fragment or epitope thereof; and an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL131A, or an antigenic fragment or epitope thereof.

[0081] In some embodiments, at least one RNA polynucleotide has an open reading frame encoding two or more HCMV antigen polypeptides, or antigenic fragments or epitopes thereof. In some embodiments, one or more open reading frames are codon-optimized. In some embodiments, at least one RNA polynucleotide is encoded by at least one nucleic acid sequence selected from SEQ ID NOs: 58, 60, 62, 64, 66, 68, and 108-113. In some embodiments, at least one of the RNA polynucleotides encodes an antigen polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the amino acid sequences of SEQ ID NOs: 59, 61, 63, 65, 67, and 69.

[0082] In some embodiments, at least one RNA polynucleotide encodes the antigenic protein of SEQ ID NO: 59, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence or 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 polynucleotide encodes the antigenic protein of SEQ ID NO: 61, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence or 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 polynucleotide encodes the antigenic protein of SEQ ID NO: 63, wherein the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence or 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 polynucleotide encodes the antigenic protein of SEQ ID NO: 65, where the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence or 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 polynucleotide encodes the antigenic protein of SEQ ID NO: 67, where the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence or 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 polynucleotide encodes the antigenic protein of SEQ ID NO: 69, where the RNA polynucleotide has less than 80% identity to the wild-type mRNA sequence or more than 80% identity to the wild-type mRNA sequence, but does not include the wild-type mRNA sequence.

[0083] In some embodiments, at least one RNA polynucleotide comprises at least one chemical modification. In some embodiments, the vaccine is multivalent. In some embodiments, the RNA polynucleotide comprises a polynucleotide sequence derived from a virus strain or isolate selected from VR1814, VR6952, VR3480B1, VR4760, Towne, TB40 / E, AD169, Merlin, and Toledo.

[0084] In some embodiments, the HCMV vaccine further comprises a second chemical modification 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.

[0085] In some embodiments, 80% of the uracils in the open reading frame have a chemical modification. 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, the chemical modification is N1-ethylpseudouridine.

[0086] In some embodiments, the vaccine is 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 ionizable 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), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). 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.

[0087] In some embodiments, the nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the nanoparticles have a net neutral charge at neutral pH. In some embodiments, the nanoparticles have an average diameter of 50 to 200 nm. Aspects of the present invention relate to methods of inducing an antigen-specific immune response in a subject, comprising administering to the subject any of the vaccines described herein in an amount effective to generate an antigen-specific immune response. In some embodiments, the antigen-specific immune response comprises a T cell response. In some embodiments, the antigen-specific immune response comprises 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 generating an antigen-specific immune response comprises a single administration of the vaccine. In some embodiments, the method further comprises administering a booster dose of the vaccine. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0088] An aspect of the invention relates to an HCMV vaccine as described herein 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 generate an antigen-specific immune response.

[0089] An aspect of the invention relates to the use of an HCMV vaccine described herein 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 generate an antigen-specific immune response.

[0090] Aspects of the invention relate to methods of preventing or treating HCMV infection, comprising administering to a subject a vaccine described herein.

[0091] Aspects of the invention relate to HCMV vaccines described herein formulated in an amount effective to generate an antigen-specific immune response in a subject. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log over a control, or by 1 to 3 logs over a control. In some embodiments, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least 2-fold over a control, at least 5-fold over a control, at least 10-fold over a control, or by 2 to 10-fold over a control.

[0092] In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject not administered an HCMV vaccine. In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject administered a live-attenuated or inactivated HCMV vaccine. In some embodiments, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject administered a recombinant or purified HCMV protein vaccine. In some embodiments, an effective amount is a dose equivalent to at least a two-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0093] In some embodiments, an effective amount is a dose equivalent to at least a four-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0094] In some embodiments, an effective amount is a dose equivalent to at least a 10-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0095] In some embodiments, an effective amount is a dose equivalent to at least a 100-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0096] In some embodiments, an effective amount is a dose equivalent to at least a 1000-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0097] In some embodiments, the effective amount is a dose equivalent to a 2- to 1000-fold reduction of a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0098] In some embodiments, the effective amount is a total dose of 50-1000 μg. In some embodiments, the effective amount is a total dose of 100 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 100 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.

[0099] In some embodiments of the methods disclosed herein, anti-HCMV antigenic polypeptide antibodies are produced in the subject and the titer of the anti-HCMV antigenic polypeptide antibodies is increased by at least 1 log relative to a control. In some embodiments, the titer of the anti-HCMV antigenic polypeptide antibodies produced in the subject is increased by 1 to 3 logs relative to a control.

[0100] In some embodiments of the methods disclosed herein, the anti-HCMV antigenic polypeptide antibody titer produced in the subject is increased by at least 2-fold relative to a control, at least 5-fold relative to a control, at least 10-fold relative to a control, or 2-10-fold relative to a control.

[0101] In some embodiments of the methods disclosed herein, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject who has not received an HCMV vaccine. In some embodiments of the methods disclosed herein, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject who has received a live-attenuated or inactivated HCMV vaccine. In some embodiments of the methods disclosed herein, the control is an anti-HCMV antigenic polypeptide antibody titer produced in a subject who has received a recombinant or purified HCMV protein vaccine.

[0102] In some embodiments of the methods disclosed herein, the effective amount is a dose equivalent to at least a two-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant HCMV protein vaccine or a live-attenuated HCMV vaccine.

[0103] In some embodiments of the methods disclosed herein, the effective amount is a dose equivalent to at least a four-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0104] In some embodiments of the methods disclosed herein, the effective amount is a dose equivalent to at least a 10-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0105] In some embodiments of the methods disclosed herein, the effective amount is a dose equivalent to at least a 100-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0106] In some embodiments of the methods disclosed herein, the effective amount is a dose equivalent to at least a 1000-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0107] In some embodiments of the methods disclosed herein, the effective amount is a dose equivalent to a 2- to 1000-fold reduction in a standard care dose of a recombinant HCMV protein vaccine, and the anti-HCMV antigenic polypeptide antibody titer produced in the subject is equivalent to the anti-HCMV antigenic polypeptide antibody titer produced in a control subject administered a standard care dose of a recombinant or purified HCMV protein vaccine or a live-attenuated or inactivated HCMV vaccine.

[0108] In some embodiments of the methods disclosed herein, the effective amount is a total dose of 50-1000 μg. In some embodiments of the methods disclosed herein, the effective amount is a total dose of 100 μg. In some embodiments of the methods disclosed herein, the effective amount is a 25 μg dose administered to the subject a total of two times. In some embodiments of the methods disclosed herein, the effective amount is a 100 μg dose administered to the subject a total of two times. In some embodiments of the methods disclosed herein, the effective amount is a 400 μg dose administered to the subject a total of two times. In some embodiments of the methods disclosed herein, the effective amount is a 500 μg dose administered to the subject a total of two times.

[0109] An embodiment of the present invention is an HCMV vaccine comprising: i) HCMV antigenic polypeptides gH, gL, UL128, UL130 and / or UL131A, or antigenic fragments or epitopes thereof; and ii) HCMV antigenic polypeptide gB, or antigenic fragments or epitopes thereof; wherein one or more of the HCMV antigenic polypeptides comprises a signal sequence linked to the HCMV antigenic polypeptide.

[0110] 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 amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 53). In some embodiments, the signal peptide is an IgGκ signal peptide. In some embodiments, the signal peptide has the amino acid sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 54). In some embodiments, the subject is a woman of childbearing age.

[0111] An embodiment of the present invention relates to a method of preventing congenital HCMV infection comprising administering to a female of childbearing age a therapeutically effective amount of a human cytomegalovirus (HCMV) vaccine comprising: i) at least one RNA polynucleotide having one or more open reading frames encoding HCMV antigenic polypeptides gH, gL, UL128, UL130 and / or UL131A, or antigenic fragments or epitopes thereof; ii) an RNA polynucleotide having an open reading frame encoding HCMV antigenic polypeptide gB, or an antigenic fragment or epitope thereof; and iv) a pharmaceutically acceptable carrier or excipient.

[0112] In some embodiments of the methods disclosed herein, the vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gH or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gL or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL128 or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL130 or an antigenic fragment or epitope thereof; and an RNA polynucleotide having an open reading frame encoding UL131 or an antigenic fragment or epitope thereof.

[0113] In some embodiments, the nucleic acid vaccines described herein are chemically modified. In other embodiments, the nucleic acid vaccines are unmodified.

[0114] Still other embodiments provide 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 polypeptide, wherein the RNA polynucleotides do not comprise a stabilizing element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

[0115] In another aspect, the invention provides compositions or methods for vaccinating a subject, the method comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first 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 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, or 80-200 μg per dose. g, 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day 0. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day 21.

[0116] In some embodiments, a 25 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. In some embodiments, a 100 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. In some embodiments, a 50 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. In some embodiments, a 75 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. In some embodiments, a 150 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. In some embodiments, a 400 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. In some embodiments, a 200 microgram dose of RNA polynucleotide is included in the nucleic acid vaccine administered to a subject. 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, and in other embodiments, the nucleic acid vaccine is not chemically modified.

[0117] Aspects of the present invention provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide (the RNA polynucleotides do not include a stabilizing element) and a pharmaceutically acceptable carrier or excipient (the vaccine does not include an adjuvant). In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence having increased GC content relative to the wild-type sequence.

[0118] Aspects of the present invention provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotides are present in a formulation for in vivo administration to a host and confer antibody titers that exceed the standard for seroprotection against the first antigen in an acceptable percentage of human subjects. In some embodiments, the antibody titers produced by the mRNA vaccines of the present invention are neutralizing antibody titers. In some embodiments, the neutralizing antibody titers are greater than those produced by protein vaccines. In other embodiments, the neutralizing antibody titers produced by the mRNA vaccines of the present invention are greater than those produced by adjuvanted protein vaccines. In still other embodiments, the neutralizing antibody titer produced by the mRNA vaccines of the present invention is 1,000-10,000, 1,200-10,000, 1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000, 2,000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500. Neutralizing titers are typically expressed as the highest serum dilution required to achieve a 50% reduction in plaque counts.

[0119] Also provided are nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotides have a stabilizing element or are formulated with an adjuvant and are present in a formulation for in vivo administration to a host to elicit a higher antibody titer that lasts longer than the antibody titer elicited by an mRNA vaccine encoding the first antigenic polypeptide. In some embodiments, the RNA polynucleotides are formulated to produce neutralizing antibodies within one week of 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.

[0120] 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 nucleotide modification, that encodes a first antigenic polypeptide, wherein the RNA polynucleotides are present in a formulation for in vivo administration to a host such that the level of antigen expression in the host significantly exceeds the level of antigen expression produced by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.

[0121] Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame containing at least one chemical modification, or optionally no nucleotide modification, encoding a first antigenic polypeptide, wherein the vaccine requires at least 10-fold less RNA polynucleotide than an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotides are present in a dose of 25-100 micrograms. Aspects of the invention also provide single-unit-use vaccines comprising between 10 μg and 400 μg of one or more RNA polynucleotides having an open reading frame containing at least one chemical modification, or optionally no nucleotide modification, encoding a first antigenic polypeptide, as well as a pharmaceutically acceptable carrier or excipient formulated for delivery to a human subject. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

[0122] Aspects of the present invention provide methods for creating, maintaining, or restoring antigenic memory to a viral strain in an individual or a population of individuals, comprising administering to the individual or population an antigenic memory booster nucleic acid vaccine comprising: (a) at least one RNA polynucleotide, such polynucleotide containing at least one chemical modification or, optionally, no nucleotide modification, and comprising two or more codon-optimized open reading frames, such open reading frames encoding a set of reference antigen polypeptides; and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual by 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.

[0123] Aspects of the present invention provide methods of vaccinating a subject, comprising administering to the subject a single dose of between 25 μg / kg and 400 μg / kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide in an amount effective to vaccinate the subject. 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 vaccine requires 10-fold less RNA polynucleotide than an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dose of 25 to 100 micrograms.

[0124] Another aspect provides a nucleic acid vaccine comprising an LNP-formulated RNA polynucleotide having an open reading frame that does not contain any nucleotide modifications (unmodified), the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10-fold less RNA polynucleotide than would be required for an unmodified mRNA vaccine not formulated in an LNP to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dose of 25-100 micrograms.

[0125] The data shown in Examples show that the immune response is significantly enhanced by using the formulation of the present invention.Surprisingly, in contrast to the reports of the prior art, it is preferable to use unchemically modified mRNA formulated in carrier for vaccine production, and it is described herein that chemically modified mRNA-LNP vaccine requires significantly lower effective mRNA dose than unmodified mRNA, that is, when formulated in carrier other than LNP, it is less than 1 / 10 of unmodified mRNA.Both the chemically modified RNA vaccine and unmodified RNA vaccine of the present invention produce better immune response than the mRNA vaccine formulated in different lipid carriers.

[0126] In other aspects, the invention includes methods of treating an elderly subject aged 60 years or older, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide, in an amount effective to vaccinate the subject. In other aspects, the invention includes methods of treating a young subject, 17 years of age or younger, comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide, in an amount effective to vaccinate the subject.

[0127] In another aspect, the invention includes a method of treating an adult subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide in an amount effective to vaccinate the subject.

[0128] In some aspects, the present invention is a method of vaccinating a subject with a combination vaccine comprising at least two nucleic acid sequences encoding antigens, wherein the vaccine is administered at a combined therapeutic dose, and wherein the dose of each individual nucleic acid encoding the antigen is subtherapeutic. In some embodiments, the combined dose is 25 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dose is 100 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dose is 50 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dose is 75 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dose is 150 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dose is 400 micrograms of RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the sub-therapeutic dose of each individual nucleic acid encoding an antigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micrograms. In other embodiments, the nucleic acid vaccine is chemically modified, and in other embodiments, the nucleic acid vaccine is not chemically modified.

[0129] In some embodiments, the RNA polynucleotide is one of SEQ ID NOs: 1-6, 58, 60, 62, 64, 66, 68, and 108-113 and comprises at least one chemical modification. In other embodiments, the RNA polynucleotide is one of SEQ ID NOs: 1-6, 58, 60, 62, 64, 66, 68, and 108-113 and does not comprise or is unmodified with any nucleotide modification. In still other embodiments, at least one RNA polynucleotide encodes an antigenic protein of any of SEQ ID NOs: 7-12, 59, 61, 63, 65, 67, and 69 and comprises at least one chemical modification. In other embodiments, the RNA polynucleotide encodes an antigenic protein of any of SEQ ID NOs: 7-12, 59, 61, 63, 65, 67, and 69 and does not comprise or is unmodified with any nucleotide modification.

[0130] In preferred embodiments, the vaccines of the present invention (e.g., LNP-encapsulated mRNA vaccines) produce 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 exemplary embodiments, antibody titer is expressed as the reciprocal of the highest dilution (in a serial dilution) that still gives a positive result. In exemplary embodiments, antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titer is determined or measured by a neutralization assay, e.g., a microneutralization assay. In certain embodiments, antibody titer measurements are expressed as a ratio, such as 1:40, 1:100, etc.

[0131] In exemplary embodiments of the invention, an effective vaccine produces antibody titers 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, antibody titers are produced or achieved by 10 days post-vaccination, by 20 days post-vaccination, by 30 days post-vaccination, by 40 days post-vaccination, or by 50 days or more post-vaccination. In exemplary embodiments, titers are produced or achieved following a single dose of vaccine administered to a subject. In other embodiments, titers are produced or achieved following multiple doses, for example, following a first and second dose (e.g., a booster dose).

[0132] In exemplary aspects of the invention, antigen-specific antibodies are measured in μg / ml, or in IU / L (International Units per Liter) or mIU / ml (milli-International Units per ml). In exemplary embodiments of the invention, an effective vaccine produces >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 produces >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 generated or achieved by 10 days after vaccination, by 20 days after vaccination, by 30 days after vaccination, by 40 days after vaccination, or by 50 days or more after vaccination. In exemplary embodiments, this level or concentration is generated or achieved after a single dose of the vaccine is administered to the subject. In other embodiments, this level or concentration is generated or achieved following multiple doses, for example, following a first and 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, for example, a microneutralization assay.

[0133] Each of the limitations of the present invention may encompass various embodiments of the present invention. Accordingly, it is anticipated that each of the limitations of the present invention, including 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 being carried out in various ways.

[0134] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like numeral. For clarity, not every component may be shown in each figure. The following are the drawings: [Brief explanation of the drawings]

[0135] [Figure 1] Figures 1A-1C show different protein complexes formed by hCMV proteins. The tropism of hCMV is dictated by distinct protein complexes. Figure 1A shows the gH / gL / gB complex that mediates hCMV entry into fibroblasts. Figure 1B shows a pentameric complex containing gH / gL / UL128 / UL130 / UL131A. Such pentameric complexes mediate hCMV entry into epithelial cells, endothelial cells, monocytes, and dendritic cells. Figure 1C is adapted from Macagno et al. (2010) J. Virology 84(2):1005-13 and shows the hCMV pentameric complex (gH / gL / UL128 / UL130 / UL131A) further complexed with antibodies specific for protein components of the pentameric complex: 8I21 (anti-pentamer), 3G16 (anti-gH), 15D8 (anti-UL128), 7I13 (anti-UL128 / UL130 / UL131A), and 10P3 (anti-gL). [Figure 2-1] Figures 2A-2D show that delivery of premixed mRNA encoding various subunits of the hCMV pentamer results in surface expression of the pentameric complex in HeLa cells. Figure 2A shows surface expression of gH. Figure 2B shows surface expression of UL128 / UL130 / UL131A. Figure 2C shows surface expression of UL128. Figure 2D shows surface expression of the pentamer. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3-1]Figures 3A-3B show that the hCMV pentamer complex is not expressed on the cell surface when one of the core subunits is absent. mRNA encoding all or part of the pentamer subunits was expressed in HeLa cells, and surface expression of the pentamer was detected using an anti-pentamer antibody (8I21). High levels of surface expression of the pentamer were detected only when all core subunits were expressed. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4] Figures 4A-B show that gH-gL dimerization is sufficient to induce surface expression of gH. Anti-gH antibody (3G16) was used to detect gH on the cell surface. When gH and gL were coexpressed, similar levels of gH were detected on the surface of HeLa cells as when all subunits in the pentameric complex were expressed. When gH was expressed alone, very little gH was detected on the surface of transfected HeLa cells. [Figure 5-1] Figures 5A-5D show the intracellular and surface expression of the hCMV antigen gB. The mRNA encoding gB was expressed both intracellularly and on the cell surface (Figures 5A-5C). Both the gB precursor and proteolytically processed mature gB were detected by immunoblotting with anti-gB antibodies (Figure 5D). An "*" indicates an overloaded lane. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6] This figure shows an immunogenicity study of the hCMV pentamer complex mRNA vaccine construct. Mice were vaccinated with the indicated doses of mRNA according to the vaccination schedule. High titers of anti-pentamer antibodies were detected in the mouse serum after immunization. Different formulations of pentamer mRNA produced comparable levels of antibodies. A third immunization did not result in a boost in antibody production. [Figure 7]This figure shows an immunogenicity study of hCMV gB mRNA vaccine constructs with or without pentameric complex mRNA constructs. The gB mRNA constructs produced similar IgG titers as the gB protein / MF59 antigen after three immunizations. A boost in IgG production was observed after the third immunization. The addition of the pentameric mRNA construct did not interfere with the induction of anti-gB IgG. [Figure 8] Neutralization studies of the hCMV pentameric complex mRNA vaccine construct in the epithelial cell line ARPE-19 are shown. IE1 staining in infected ARPE-19 cells is demonstrated. Immunization with the hCMV pentameric complex mRNA vaccine construct elicits highly potent neutralizing antibodies in mice. The neutralizing antibody titer (1:25600) in mouse serum on day 41 (3 weeks after the second immunization) was capable of neutralizing the hCMV clinical isolate VR1814 in ARPE-19 cells. [Figure 9] Measurement of hCMV neutralizing IgG titers in ARPE-19 cells infected with hCMV clinical isolate VR1814. See also Table 5. [Figure 10-1] Figures 10-10B show surface expression in HeLa cells of the hCMV pentameric complex (gH-gL-UL128-UL130-UL131A) encoded by the first-generation pentameric construct described herein (referred to as "old") and the second-generation pentameric construct (referred to as "new") also described herein. The sequences of the mRNAs within the second-generation constructs are provided in Table 6, corresponding to SEQ ID NOS: 58-69. Figures 10A and 10C show the results of a fluorescence-activated cell sorting (FACS) experiment using the 8I21 (anti-pentamer) antibody to detect surface expression of the pentameric complex. Surface expression of the pentameric complex is indicated by the emergent fluorescent cell population. Figures 10B and 10D show quantification of the FACS experiment. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11]Figures 11A-11E show Western blots showing the expression of subunits of the hCMV pentameric complex (gH, gL, UL128, UL130, and UL131A) encoded by the first-generation pentameric construct described herein (referred to as "old") and the second-generation pentameric construct (referred to as "new") also described herein. [Figure 12] Figure 12 shows that immunization with the pentamer mRNA complex elicits high antibody titers that are maintained for up to several months. Immunogenicity studies of the hCMV pentamer complex mRNA vaccine construct are shown. Balb / c mice were vaccinated with the indicated doses of mRNA (lower panel) according to the vaccination schedule. Mouse serum IgG titers were measured 20, 41, 62, 92, and 123 days after immunization. Serum IgG titers were measured using hCMV pentamer-coated plates. High titers of anti-pentamer antibodies were detected in the serum of immunized mice. [Figure 13] This shows that the hCMV mRNA vaccine construct induced higher neutralizing antibody titers in mice than CytoGam®, a hyperimmune serum used clinically for the prevention of hCMV. Balb / c mice were vaccinated according to the vaccination schedule with the indicated doses of mRNA (lower panel). Neutralizing antibody titers in mouse serum were measured in ARPE-19 epithelial cells infected with hCMV clinical isolate VR1814 on days 42, 122, 152, and 182 post-immunization. The high titers of neutralizing antibodies induced by the hCMV pentamer complex mRNA vaccine were maintained for up to 6 months. [Figure 14] This graph shows the neutralizing antibody titers induced in mice by the hCMV pentamer complex mRNA vaccine construct. Balb / c mice were vaccinated according to the vaccination schedule with the indicated dose of mRNA (lower panel). Neutralizing antibody titers in mouse serum were measured 42, 62, and 182 days after immunization using HEL299 fibroblasts infected with 500-2000 pfu of hCMV AD169 strain. [Figure 15]Schematic diagram of pentameric subunits linked by a self-cleaving 2A peptide (e.g., as described in Kim et al., PLoS ONE 6(4):e18556, 2011). [Figure 16] 1 is a Western blot showing that gH and gL linked by the 2A peptide undergo efficient autocleavage to generate individual gH and gL subunits. [Figure 17] 1 is a graph showing that individual gH and gL subunits generated from autocleavage of the linked 2A peptide were able to dimerize and translocate to the cell surface. [Figure 18] Figures 18A-18B show high and durable titers of anti-pentamer binding and neutralizing antibodies in mice. Figure 18A shows a graph depicting anti-pentamer antibody titers. Equimolar and equal mass preparations of pentamer mRNA were compared and found to be equally effective. Figure 18B shows a graph depicting neutralization titers measured in ARPE19 epithelial cells infected with hCMV strain VR1814. Equimolar and equal mass preparations of pentamer mRNA were compared and found to be equally effective. Neutralization titers were found to be approximately 25-fold higher than CytoGam®. [Figure 19] Figures 19A-19C show that neutralizing activity against epithelial cell infection is dependent on anti-pentamer antibodies. Figure 19A shows that the depleted proteins were either pentamers or gH / gL dimers. Figures 19B and 19C show graphs demonstrating neutralization. Figure 19B shows neutralization by sera from mice immunized with pentamers or gH / gL dimers. Figure 19C shows neutralization by CytoGam® in combination with pentamers or gH / gL. [Figure 20]Figures 20A-20B are graphs showing the immunogenicity of second-generation hCMV mRNA vaccine constructs formulated with Compound 25 lipid. Second-generation mRNA constructs encoding the pentamer and gB induced pentamer-specific (Figure 20A) and gB-specific (Figure 20B) antibodies as early as 20 days after the initial immunization. Pentamer- and gB-specific antibody titers continued to increase in mice after booster doses. [Figure 21] 1 is a graph showing that a total dose of 3 μg of an HCMV mRNA vaccine construct encoding a pentameric complex induces higher neutralizing antibody titers than CytoGam®, a hyperimmune serum used clinically for the prevention of hCMV. DETAILED DESCRIPTION OF THE INVENTION

[0136] Embodiments of the present disclosure provide an RNA (e.g., mRNA) vaccine containing a polynucleotide encoding a human cytomegalovirus (HCMV) antigen. Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virus belonging to the Herpesviridae family. HCMV consists of a DNA core, an outer capsid, and a lipid membrane (envelope) incorporating virus-specific glycoproteins. Its diameter is approximately 150-200 nm. The genome is linear and non-segmented, approximately 200 kb in length. Viral replication is nuclear and lysogenic. Replication is by bidirectional dsDNA replication.

[0137] HCMV can infect a wide range of mammalian cells, which correlates with its ability to infect most organs and tissues. Entry into host cells is achieved by the attachment of viral glycoproteins to host cell receptors that mediate endocytosis. HCMV exhibits a broad host cell range, capable of infecting several cell types, including endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, leukocytes, and dendritic cells. This broad cell tropism suggests that HCMV may bind to multiple receptors or common surface molecules. The HCMV coat is highly complex, containing more than 20 glycoproteins, which may explain the broad cell tropism of HCMV. HCMV particles contain at least four major glycoprotein complexes, all of which are involved in HCMV infection, which requires initial interaction with the cell surface through binding to heparin sulfate proteoglycans and possibly other surface receptors.

[0138] The gCI complex consists of a dimeric molecule of glycoprotein gB. Each 160 kDa monomer is cleaved to generate a 116 kDa surface unit linked by a disulfide bond to a 55 kDa transmembrane component. Some antibodies immunospecific for gB inhibit virion attachment to cells, whereas others block fusion of infected cells, suggesting that the gB protein may exert multiple functions at the initiation of infection. Studies have confirmed that glycoprotein B (gB) facilitates HCMV entry into cells by binding to receptors and mediating membrane fusion. Several cell membrane proteins interact with gB, and this interaction likely facilitates entry and activates cell signaling pathways.

[0139] The gCII complex is the most abundant glycoprotein complex and is a heterodimer consisting of glycoproteins gM and gN. This complex binds to heparan sulfate proteoglycans, suggesting that it may contribute to the initial interaction of the virion with the cell surface. Similar to the gM-gN complex found in some α-herpesviruses, it may also play a structural role during virion assembly / coating.

[0140] The gCIII complex is a trimer consisting of glycoproteins gH, gL, and gO covalently linked by disulfide bonds. All known herpesviruses encode a gH-gL heterodimer that mediates fusion of the virion envelope with the cell membrane. Antibodies specific for human CMV gH do not affect viral attachment but block penetration and cell-to-cell transmission. A gO-deficient mutant of HCMV (strain AD169) exhibits a significant growth defect.

[0141] The HCMV proteins UL128, UL130, and UL131A associate with the gH and gL proteins to form a heteropentameric complex (designated gH / gL / UL128-131A) found on the surface of HCMV. Natural variants, as well as deletion and mutation analyses, have implicated proteins of the gH / gL / UL128-131A complex in the ability to infect specific cell types, including, for example, endothelial cells, epithelial cells, and leukocytes.

[0142] HCMV enters cells by fusing its envelope with either the plasma membrane (fibroblasts) or the endosomal membrane (epithelial and endothelial cells). HCMV initiates cell entry by binding to cell surface heparan sulfate proteoglycans using the envelope glycoprotein M (gM) or gB. This step is followed by interaction with cell surface receptors that trigger or initiate intracellular signaling for entry. Entry receptor function is provided by the gH / gL glycoprotein complex. Different gH / gL complexes are known to facilitate entry into epithelial, endothelial, or fibroblast cells. For example, entry into fibroblasts requires the gH / gL heterodimer, whereas entry into epithelial and endothelial cells requires the pentameric complex gH / gL / UL128 / UL130 / UL131 in addition to gH / gL. Thus, different gH / gL complexes engage different entry receptors on epithelial / endothelial cells and fibroblasts. Receptor engagement is followed by membrane fusion, a process mediated by gB and gH / gL. Early antibody studies supported a critical role for both gB and gH / gL in HCMV entry. gB is essential for entry and cell spread. gB and gH / gL are necessary and sufficient for cell fusion and thus constitute the HCMV "fusion machinery" that is conserved among other herpesviruses.

[0143] Thus, the four glycoprotein complexes play crucial roles in viral attachment, binding, fusion and entry into host cells.

[0144] Studies involving the gH / gL / UL128-131A complex have demonstrated that HCMV glycoproteins gB, gH, gL, gM, and gN, as well as UL128, UL130, and UL131A proteins, are antigenic and participate in immune stimulatory responses in various cell types. Furthermore, the UL128, UL130, and UL131A genes are relatively conserved among HCMV isolates and are therefore attractive targets for vaccination. Furthermore, recent studies have shown that antibodies directed against epitopes within the pentameric gH / gL / UL128-131 complex neutralize entry into endothelial, epithelial, and other cell types, thus blocking HCMV's ability to infect several cell types.

[0145] The HCMV envelope glycoprotein complex (gCI, II, III, gH / gL / UL128-131A) is the primary antigenic target of the antiviral immune response. Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines comprising polynucleotides encoding HCMV antigens, particularly HCMV antigens derived from one of the HCMV glycoprotein complexes. Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines comprising at least one polynucleotide encoding at least one HCMV antigen polypeptide. The HCMV 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.

[0146] The entire contents of International Application No. PCT / US2015 / 027400 (WO2015 / 164674), entitled "Nucleic Acid Vaccines," are incorporated herein by reference. The mRNA vaccines described herein have been discovered to be superior to current vaccines in several ways. First, lipid nanoparticle (LNP) delivery is superior to other formulations, including protamine-based approaches described in the literature, and no additional adjuvants are required. The use of LNPs allows for the effective delivery of chemically modified or unmodified mRNA vaccines. Furthermore, both modified and unmodified LNP-formulated mRNA vaccines have been demonstrated 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.

[0147] While attempts have been made to produce functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic efficacy of these RNA vaccines has not yet been fully established. Quite surprisingly, the inventors have discovered, through embodiments of the present invention, a class of formulations for in vivo delivery of mRNA vaccines that produce significantly enhanced and in many respects synergistic immune responses, including enhanced antigen production and functional antibody production with neutralizing capacity. These results can be achieved even when significantly lower doses of mRNA are administered compared to the mRNA doses used in other classes of lipid-based formulations. The formulations of the present invention have demonstrated significant and unexpected 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 generate an immunogenic response. The formulations of the present invention do not require viral replication to produce sufficient protein to produce a potent immune response. Therefore, the mRNA of the present invention is not self-replicating RNA and does not contain components necessary for viral replication.

[0148] In some embodiments, the present invention relates to the surprising discovery that lipid nanoparticle (LNP) formulation significantly improves the efficacy of mRNA vaccines, including chemically modified mRNA vaccines and unmodified mRNA vaccines.The efficacy of mRNA vaccines formulated in LNPs has been tested in vivo using several different antigens.The results presented herein show the unexpected superior efficacy of mRNA vaccines formulated in LNPs compared to other commercially available vaccines.

[0149] In addition to providing an enhanced immune response, the formulations of the present invention generate a more rapid immune response with a smaller dose of antigen than other vaccines tested. The mRNA-LNP formulations of the present invention also produce quantitatively and qualitatively superior immune responses than vaccines formulated in different carriers. The data presented herein demonstrate that the formulations of the present invention provide significant and unexpected improvements over existing antigen vaccines. Furthermore, the mRNA-LNP formulations of the present invention outperform other vaccines, even when the mRNA dose is lower than that of other vaccines.

[0150] The LNPs used in the studies described herein have previously been used to deliver siRNA in various animal models as well as in humans. In light of the observations made in relation to siRNA delivery using LNP formulations, the fact that LNPs are useful for vaccines is quite surprising. It has been observed that therapeutic delivery of siRNA formulated in LNPs causes undesirable inflammatory reactions associated with transient IgM responses, typically resulting in reduced antigen production and a diminished immune response. In contrast to the findings observed with siRNA, the LNP-mRNA formulations of the present invention have been demonstrated herein to generate enhanced IgG levels sufficient for prophylactic and therapeutic methods, rather than a transient IgM response.

[0151] Nucleic Acids / Polynucleotides The human cytomegalovirus (HCMV) vaccines provided herein comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV 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.

[0152] In some embodiments, at least one RNA polynucleotide of the HCMV vaccine is encoded by at least one nucleic acid sequence selected from any of SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, and 68. In some embodiments, at least one RNA polynucleotide of the HCMV vaccine is encoded by at least one fragment of a nucleic acid sequence selected from any of SEQ ID NOs: 1-31, 58, 60, 62, 64, 66, and 68.

[0153] In some embodiments, the RNA vaccine comprises an RNA polynucleotide having an open reading frame encoded by SEQ ID NO: 58 or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoded by SEQ ID NO: 60 or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoded by SEQ ID NO: 62 or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoded by SEQ ID NO: 64 or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoded by SEQ ID NO: 66 or an antigenic fragment or epitope thereof, and an RNA polynucleotide having an open reading frame encoded by SEQ ID NO: 68 or an antigenic fragment or epitope thereof.

[0154] The nucleic acid (also called polynucleotide) may be or include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, LNA with a β-D-ribo configuration, α-LNA with an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0155] 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 (naturally occurring, non-naturally occurring, or modified polymer of amino acids), which can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. In some preferred embodiments, mRNA is translated in vivo. Those skilled in the art will understand that, unless otherwise noted, the polynucleotide sequences described in this application list a "T" in a representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" is replaced with a "U." Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, with each "T" in the DNA sequence replaced with a "U."

[0156] Typically, the basic components of an mRNA molecule 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 may function as mRNAs, but may be distinguished from wild-type mRNAs in their functional and / or structural design features that help overcome existing problems in effective polypeptide expression using nucleic acid-based therapeutics.

[0157] Some embodiments of the present disclosure provide HCMV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or immunogenic fragment or epitope thereof. Some embodiments of the present disclosure provide HCMV vaccines comprising at least one RNA polynucleotide having an open reading frame encoding two or more HCMV antigen polypeptides or immunogenic fragments or epitopes thereof. Some embodiments of the present disclosure provide HCMV vaccines comprising two or more RNA polynucleotides having open reading frames encoding two or more HCMV antigen polypeptides or immunogenic fragments or epitopes thereof. The one or more HCMV antigen polypeptides may be encoded on a single RNA polynucleotide or may be individually encoded by multiple (e.g., two or more) RNA polynucleotides.

[0158] Some embodiments of the present disclosure provide HCMV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having a single open reading frame encoding two or more (e.g., two, three, four, five, or more) HCMV antigen polypeptides or immunogenic fragments or epitopes thereof. Some embodiments of the present disclosure provide HCMV vaccines comprising at least one ribonucleic acid (RNA) polynucleotide having two or more open reading frames, e.g., two, three, four, five, or more open reading frames, encoding two, three, four, five, or more HCMV antigen polypeptides. In any of these embodiments, the at least one RNA polynucleotide may encode two or more HCMV antigen polypeptides selected from gH, gB, gL, gO, gM, gN, UL83, UL123, UL128, UL130, UL131A, and fragments or epitopes thereof. In some embodiments, the at least one RNA polynucleotide encodes UL83 and UL123. In some embodiments, at least one RNA polynucleotide encodes gH and gL. In some embodiments, at least one RNA polynucleotide encodes UL128, UL130, and UL131A. In some embodiments, at least one RNA polynucleotide encodes gH, gL, UL128, UL130, and UL131A.

[0159] In some embodiments, the vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gH, or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gL, or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL128, or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL130, or an antigenic fragment or epitope thereof; an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide UL131A, or an antigenic fragment or epitope thereof; and an RNA polynucleotide having an open reading frame encoding the HCMV antigen polypeptide gB, or an antigenic fragment or epitope thereof.

[0160] In some embodiments, in which at least one RNA polynucleotide has a single open reading frame encoding two or more (e.g., 2, 3, 4, 5, or more) HCMV antigen polypeptides, the RNA polynucleotide may further comprise additional sequences, e.g., linker sequences, or sequences that aid in processing of the HCMV RNA transcript or polypeptide, e.g., cleavage site sequences. In some embodiments, the additional sequence may be a protease sequence, such as a furin sequence. Furin, also known as PACE (paired basic amino acid cleaving enzyme), is a calcium-dependent serine endoprotease that cleaves precursor proteins at paired basic amino acid processing sites into biologically active products. Some of its substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, probeta-secretase, membrane type 1 matrix metalloproteinase, the beta subunit of nerve growth promoting factor, and von Willebrand factor. While certain viral envelope proteins must be cleaved by furin to be fully functional, some viruses require furin processing during host cell entry. T cells require furin to maintain peripheral immune tolerance. In some embodiments, the additional sequence may be a self-cleaving 2A peptide, such as the P2A, E2A, F2A, or T2A sequence. In some embodiments, a linker sequence and a cleavage site sequence are interspersed between sequences encoding HCMV polypeptides. 2A peptides are small, "self-cleaving" peptides approximately 18-22 amino acids in length. The ribosome skips synthesis of the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, resulting in cleavage of the 2A peptide and the peptide immediately downstream. They are frequently used in biomedical research to simultaneously express two or more genes in cells using a single plasmid. Many 2A peptides exist, including those from foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), porcine teschovirus-1 2A (P2A), and thoseaasigna virus 2A (T2A).T2A has the highest cleavage efficiency (close to 100%), followed by E2A, P2A and F2A. The amino acid sequences are as follows: P2A: (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 70); T2A: (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 71); E2A: (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 72); F2A: (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:73). In some embodiments, the linker sequence and cleavage site sequence are interspersed between the sequences encoding the HCMV polypeptides. In some embodiments, the RNA polynucleotide is encoded by SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0161] In some embodiments, the RNA polynucleotide of the HCMV vaccine encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the HCMV vaccine encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the HCMV vaccine encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, the RNA polynucleotide of the HCMV 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. In some embodiments, the polynucleotides of the present disclosure are codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. In some embodiments, codon optimization may be used to match the codon frequency in the target organism and the host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites in the encoded protein (e.g., glycosylation sites); add, remove, or shuffle protein domains; insert or remove restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate to allow various domains of the protein to fold properly; or reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art - non-limiting examples of which include services by GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0162] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring 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 a naturally occurring or wild-type sequence (e.g., a naturally occurring 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 95% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring 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 with a naturally occurring or wild-type sequence (e.g., a naturally occurring 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 with a naturally occurring or wild-type sequence (e.g., a naturally occurring 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 a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide).

[0163] 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 naturally occurring or wild-type sequence (e.g., a naturally occurring 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 65% to 75% or about 80% sequence identity to the naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0164] Those skilled in the art will understand that unless otherwise specified, the polynucleotide sequences set forth in this application will list "T" in a representative DNA sequence, but if the sequence is RNA, then "T" is replaced with "U."

[0165] Antigens / antigenic polypeptides In some embodiments, the antigenic polypeptide is an HCMV glycoprotein. For example, the HCMV glycoprotein can be HCMV gB, gH, gL, gO, gN, or gM, or an immunogenic fragment or epitope thereof. In some embodiments, the antigenic polypeptide is an HCMV gH polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gL polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gB polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gO polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gN polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gM polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gC polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gN polypeptide. In some embodiments, the antigenic polypeptide is an HCMV gM polypeptide.

[0166] In some embodiments, the antigenic polypeptide is an HCMV protein or immunogenic fragment or epitope thereof selected from UL83, UL123, UL128, UL130, and UL131A. In some embodiments, the antigenic polypeptide is an HCMV UL83 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL123 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL128 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL130 polypeptide. In some embodiments, the antigenic polypeptide is an HCMV UL131A polypeptide.

[0167] In some embodiments, the antigenic HCMV polypeptide comprises two or more HCMV polypeptides. The two or more HCMV polypeptides may be encoded by a single RNA polynucleotide or by two or more RNA polynucleotides, for example, each glycoprotein may be encoded by a separate RNA polynucleotide. In some embodiments, the two or more HCMV polypeptides may be any combination of HCMV gH, gL, gB, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides may be any combination of polypeptides selected from HCMV gH, and gL, gB, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides may be any combination of HCMV gB and a polypeptide selected from HCMV gH, gL, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides may be any combination of HCMV gL and a polypeptide selected from HCMV gH, gB, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides may be any combination of HCMV gH, gL and a polypeptide selected from HCMV gB, gO, gN, gM, UL83, UL123, UL128, UL130, and UL131A polypeptides, or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides may be any combination of HCMV gH, gL 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 HCMV polypeptides may be any combination of HCMV gH, gL, and a polypeptide selected from UL83, UL123, UL128, UL130, and UL131A polypeptides or immunogenic fragments or epitopes thereof. In some embodiments, the two or more HCMV polypeptides are UL128, UL130, and UL131A. In some embodiments, the two or more HCMV polypeptides are gH and gL. In some embodiments, the two or more HCMV polypeptides are gH, gL, UL128, UL130, and UL131A. In some embodiments, the two or more HCMV polypeptides are gB, gH, gL, UL128, UL130, and UL131A.

[0168] The present disclosure includes mutant HCMV antigen polypeptides. In some embodiments, the mutant HCMV antigen polypeptide is a mutant HCMV gH polypeptide. In some embodiments, the mutant HCMV antigen polypeptide is a mutant HCMV gL polypeptide. In some embodiments, the mutant HCMV antigen polypeptide is a mutant HCMV gB polypeptide. The mutant HCMV polypeptide is designed to facilitate passage of the antigen polypeptide through the ER / Golgi, resulting in increased surface expression of the antigen. In some embodiments, the mutant HCMV polypeptide is truncated to delete one or more of the following domains: the hydrophobic membrane proximal domain, the transmembrane domain, and the cytoplasmic domain. In some embodiments, the mutant HCMV polypeptide is truncated to include only the extracellular domain sequence. For example, the mutant HCMV polypeptide may be a truncated HCMV gH polypeptide, a truncated HCMV gB polypeptide, or a truncated HCMV gL polypeptide comprising at least amino acids 1-124, including, for example, 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, as well as polypeptide fragments having fragment sizes within the recited size ranges.

[0169] In some embodiments, the HCMV antigen 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 or multi-chain polypeptides, such as antibodies or insulin, and may be associated or linked. Disulfide bonds are most commonly found in multivalent 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. The term "polypeptide variant" refers to molecules 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 share at least 50% identity with the native or reference sequence. In some embodiments, variants share at least 80% or at least 90% identity with the native or reference sequence.

[0170] In some embodiments, "mutant mimics" are provided. As used herein, the term "mutant mimics" refers to those that contain at least one amino acid that mimics an activation sequence. For example, glutamate can serve as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, mutant mimics can cause inactivation or produce inactivated products containing mimics; for example, phenylalanine can act as an inactivating substitute for tyrosine; or alanine can act as an inactivating substitute for serine.

[0171] "Orthologs" refer to genes in different species that have evolved from a common ancestral gene through speciation. Orthologs usually retain the same function throughout evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes.

[0172] By "analog" it is meant to include polypeptide variants that differ by one or more amino acid changes, such as substitutions, additions or deletions of amino acid residues, that still retain one or more properties of the parent or starting polypeptide.

[0173] The present disclosure provides several types of polynucleotide- or polypeptide-based compositions, including variants and derivatives. These include, for example, substitution, insertion, deletion, 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 altered in any way relative to a reference or starting molecule. 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 amino acid, such as one or more lysines, may be added to a peptide sequence (e.g., at the N- or C-terminus). The sequence tag may be used for peptide detection, purification, or localization. Lysines may be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of a peptide or protein amino acid sequence may be deleted as needed to obtain a truncated sequence. Specific amino acids (e.g., C- or N-terminal residues) may alternatively be deleted depending on the use of the sequence, for example, for expression of the sequence as part of a larger sequence that is soluble or linked to a solid support.

[0174] "Substitutional variants," when referring to polypeptides, are those in which at least one amino acid residue in a native or starting sequence has been removed and a different amino acid inserted in its place at 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.

[0175] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with an amino acid of a different size, charge, or polarity. Examples of conservative substitutions include the substitution of non-polar (hydrophobic) residues such as isoleucine, valine, and leucine for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another, are further examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, and methionine for polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue with a non-polar residue.

[0176] A "feature," when referring to a polypeptide or polynucleotide, is defined as a distinct amino acid sequence-based or nucleotide-based component of a molecule, respectively. A feature of a polypeptide encoded by a polynucleotide can include a surface manifestation, a local conformational shape, a fold, a loop, a half-loop, a domain, a half-domain, a site, an end, or any combination thereof.

[0177] As used herein, the term "domain," when referring to a polypeptide, refers to a polypeptide motif having one or more identifiable structural or functional features or characteristics (e.g., binding ability to serve as a site of protein-protein interaction). As used herein when referring to a polypeptide, the term "site" in relation to amino acid-based embodiments is used synonymously with "amino acid residue" and "amino acid side chain." As used herein, the term "site" in relation to polynucleotides, when it pertains to nucleotide-based embodiments, is used synonymously with "nucleotide." 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.

[0178] As used herein, when referring to a polypeptide or polynucleotide, the term "terminus(s)" refers to the terminus(s) of the polypeptide or polynucleotide, respectively. Such termini may refer not only to the first or last site of the polypeptide or polynucleotide, but also to additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules can be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are sometimes composed of multiple polypeptide chains held together by disulfide bonds or non-covalent forces (multimers, oligomers). These proteins have multiple N-termini and C-termini. Alternatively, the termini of a polypeptide may sometimes be modified to begin or end with a non-polypeptide-based moiety (e.g., an organic conjugate).

[0179] As will be appreciated 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, provided herein is any protein fragment that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length (meaning a polypeptide sequence that is shorter than the reference polypeptide sequence but otherwise identical to at least one amino acid residue). In another example, any protein that contains 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 shown in any of the sequences provided or referenced herein.

[0180] A polypeptide or polynucleotide molecule of the present disclosure may share a degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or polynucleotide), for example, by 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 means the degree of sequence relatedness between two or more strings of amino acid or nucleic acid residues, as determined by the number of matches between them. Identity measures the percent of identical matches between two or more sequences, with gap alignment (if any) accommodated 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 polypeptide or polynucleotide sequences, 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 introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art.Identity is based on the calculation of identity percentage, but it is understood that the value may differ due to the gap and penalty introduced in the calculation.Generally, the variant of specific polynucleotide or polypeptide has 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 with its specific reference polynucleotide or polypeptide, as determined by the sequence alignment program and parameters described herein and known to those skilled in the art.Tools for such alignment 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 common local alignment technique 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 technique 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 intentionally generates global alignments of nucleotide and protein sequences more quickly than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0181] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (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, polymer molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 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 stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if they are at least 50%, 60%, 70%, 80%, or 90% identical over at least one stretch of at least 20 amino acids.

[0182] Homology means that the compared sequences have diverged from a common origin in evolution. 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 derived from a common ancestral sequence. The term "homolog" can apply to the relationship between genes and / or proteins separated by a speciation event or by a gene duplication event. An "ortholog" is a gene (or protein) from a different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function during evolution. A "paralog" is a gene (or protein) related by duplication within a genome. Orthologs retain the same function during evolution, while paralogs evolve new functions while related to the original.

[0183] The term "identity" refers to the overall relatedness between polymer molecules, for example, between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percent identity of two polynucleic acid sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 (each of which is incorporated herein by reference). For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, the method disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs.Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG program package, Devereux, J. et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0184] In some embodiments, the polypeptide further comprises additional sequences or functional domains. For example, the HCMV polypeptides of the present disclosure may comprise one or more linker sequences. In some embodiments, the HCMV of the present invention may comprise a polypeptide tag, for example, an affinity tag (chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), SBP-tag, Strep-tag, AviTag, calmodulin-tag); a solubilization tag; a chromatography tag (a polyanionic amino acid tag such as a FLAG tag); an epitope tag (a short peptide sequence that binds to a high-affinity antibody, such as a V5 tag, Myc tag, VSV tag, Xpress tag, E tag, S tag, and HA tag); or a fluorescent tag (e.g., GFP). In some embodiments, the HCMV of the present invention may comprise an amino acid tag, such as one or more lysines, histidines, or glutamic acids, that can be added to the polypeptide sequence (e.g., at the N-terminus or C-terminus). Lysines may be used to increase peptide solubility or to enable biotinylation. Protein and amino acid tags are peptide sequences that are genetically grafted onto recombinant proteins.Sequence tags are attached to proteins for various purposes, such as peptide purification, identification or localization, for use in various applications, including affinity purification, protein arrays, Western blotting, immunofluorescence and immunoprecipitation.These tags can then be removed by chemical agents or by enzymatic means, such as specific proteolysis or intein splicing. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein may be deleted as needed to provide a truncated sequence. Specific amino acids (e.g., C- or N-terminal residues) may alternatively be deleted depending on the use of the sequence, for example, expression of the sequence as part of a larger sequence that is soluble or solid-bound.

[0185] Multiprotein and multicomponent vaccines The present disclosure encompasses HCMV vaccines, e.g., vaccines against human cytomegalovirus that include multiple RNA (e.g., mRNA) polynucleotides, each encoding a single antigenic polypeptide, and HCMV vaccines that include a single RNA polynucleotide encoding two or more antigenic polypeptides (e.g., as a fusion polypeptide). Thus, a vaccine composition that includes an RNA polynucleotide having an open reading frame encoding a first HCMV antigenic polypeptide and an RNA polynucleotide having an open reading frame encoding a second HCMV antigenic polypeptide should be understood to include (a) vaccines that include a first RNA polynucleotide encoding the first HCMV antigenic polypeptide and a second RNA polynucleotide encoding the second HCMV antigenic polypeptide, and (b) vaccines that include a single RNA polynucleotide encoding the first and second HCMV antigenic polypeptides (e.g., as a fusion polypeptide). In some embodiments, the HCMV RNA vaccines of the present disclosure 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 HCMV antigenic polypeptide (or a single RNA polynucleotide encoding two to ten or more different HCMV antigenic polypeptides). In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding an HCMV glycoprotein.In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding HCMV glycoprotein B (gB), an RNA polynucleotide having an open reading frame encoding HCMV glycoprotein M (gM), an RNA polynucleotide having an open reading frame encoding HCMV glycoprotein N (gN), an RNA polynucleotide having an open reading frame encoding HCMV glycoprotein H (gH), an RNA polynucleotide having an open reading frame encoding HCMV glycoprotein L (gL), and an RNA polynucleotide having an open reading frame encoding HCMV glycoprotein O (gO). In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV gB protein. In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV UL128 protein. In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV UL130 protein. In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV UL131 protein. In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV gM and gN proteins. In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV gH, gL, and gO proteins. In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding the HCMV gH, gL, UL128, UL130, and UL131A proteins.In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having one or more open reading frames encoding the HCMV UL83, UL128, UL123, UL130, or UL131A protein. In some embodiments, the HCMV RNA vaccine further comprises an RNA polynucleotide having an open reading frame encoding one or more (e.g., 2, 3, 4, 5, 6, or 7) HCMV proteins.

[0186] In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having one or more open reading frames encoding HCMV gH, gL, UL128, UL130, and UL131A proteins, or fragments thereof, and HCMV gB protein, or fragments thereof.

[0187] In some embodiments, the HCMV RNA vaccine comprises an RNA polynucleotide having an open reading frame encoding a gH protein or a fragment thereof, an RNA polynucleotide having an open reading frame encoding a gL protein or a fragment thereof, an RNA polynucleotide having an open reading frame encoding a UL128 protein or a fragment thereof, an RNA polynucleotide having an open reading frame encoding a UL130 protein or a fragment thereof, an RNA polynucleotide having an open reading frame encoding a UL131A protein or a fragment thereof, and an RNA polynucleotide having an open reading frame encoding a gB protein or a fragment thereof. In some embodiments, the RNA polynucleotide encodes an HCMV antigen polypeptide fused to a signal peptide (e.g., SEQ ID NO: 53 or 54). The signal peptide may be fused at the N-terminus or C-terminus of the antigen polypeptide.

[0188] signal peptide In some embodiments, the antigenic polypeptide encoded by the HCMV nucleic acid contains a signal peptide. Signal peptides, which comprise the N-terminal 15 to 60 amino acids of a protein, are typically required for translocation across membranes in the secretory pathway and thus universally control entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. Signal peptides generally contain N-terminal regions of varying lengths, usually containing three regions: 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 growing peptide chain across it. However, the signal peptide does not contribute to the final destination of the mature protein. Secretory proteins lacking additional addressing tags in their sequence are secreted into the external environment by default. The signal peptide is either cleaved from the precursor protein by signal peptidases present in the endoplasmic reticulum (ER) or remains uncleaved and functions as a membrane anchor. In recent years, a more sophisticated view of signal peptides has evolved, showing that the function and immunodominance of particular signal peptides is much more versatile than previously anticipated.

[0189] The HCMV vaccine of the present disclosure may, for example, comprise an RNA polynucleotide encoding an artificial signal peptide, wherein the signal peptide coding sequence is operably linked to and in-frame with the coding sequence of an HCMV antigen polypeptide. Thus, in some embodiments, the HCMV vaccine of the present disclosure produces an antigen polypeptide comprising an HCMV antigen polypeptide fused to a signal peptide. In some embodiments, the signal peptide is fused to the N-terminus of the HCMV antigen polypeptide. In some embodiments, the signal peptide is fused to the C-terminus of the HCMV antigen polypeptide.

[0190] In some embodiments, the signal peptide fused to the HCMV antigen polypeptide is an artificial signal peptide. In some embodiments, the artificial signal peptide fused to the HCMV antigen polypeptide encoded by the HCMV RNA vaccine is derived from an immunoglobulin protein, for example, an IgE signal peptide or an IgG signal peptide. In some embodiments, the signal peptide fused to the HCMV antigen polypeptide encoded by the HCMV mRNA vaccine is an Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the following sequence: MDWTWILFLVAAATRVHS (SEQ ID NO: 53). In some embodiments, the signal peptide fused to the HCMV antigen polypeptide encoded by the HCMV RNA vaccine is an IgG signal peptide having the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 54). k Chain V-III region HAH signal peptide (IgG k SP). In some embodiments, the signal peptide fused to the HCMV antigen polypeptide encoded by the HCMV RNA vaccine has the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54. The examples disclosed herein are not meant to be limiting, and any signal peptide known in the art to facilitate targeting of proteins to the ER for processing and / or targeting of proteins to the cell membrane may be used in accordance with the present disclosure.

[0191] The signal peptide may have a length of 15 to 60 amino acids. For example, the signal peptide may have a length of 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 some embodiments, the signal peptide is 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-55 It may have a length of 0, 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.

[0192] Non-limiting examples of HCMV antigen polypeptides fused to signal peptides encoded by the HCMV RNA vaccines of the present disclosure can be found in Table 2, SEQ ID NOs: 32-52.

[0193] The signal peptide is typically cleaved from the nascent polypeptide at the cleavage junction during ER processing. The mature HCMV antigen polypeptides produced by the HCMV RNA vaccines of the present disclosure typically do not include the signal peptide.

[0194] chemical modification The HCMV RNA 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 HCMV antigen polypeptide or immunogenic fragment thereof, comprising at least one chemical modification.

[0195] The terms "chemical modification" and "chemically modified" refer to modifications of 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 of the 5'-terminal mRNA cap portion. With respect to polypeptides, the term "modification" refers to modifications to the canonical set of 20 amino acids. The polypeptides provided herein are also considered "modified" of those containing amino acid substitutions, insertions, or a combination of substitutions and insertions.

[0196] A polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide), in some embodiments, comprises a variety of (two or more) different modifications. In some embodiments, specific regions of a polynucleotide comprise one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or organism, respectively, exhibits reduced degradation in the cell or organism compared to a non-modified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or organism, respectively, may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively.

[0197] Modifications of polynucleotides include, but are not limited to, those described herein. Polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides) can contain naturally occurring or non-naturally occurring modifications, or polynucleotides can contain a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides can contain any useful modification, for example, of the sugar, nucleobase, or internucleoside linkage (e.g., to the linking phosphate group, phosphodiester bond, or phosphodiester backbone).

[0198] Polynucleotides (e.g., RNA polynucleotides, e.g., 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. The modifications can be in the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced by chemical synthesis or using polymerase enzymes at the end of the chain or elsewhere in the chain. Any region of the polynucleotide can be chemically modified.

[0199] The present disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides). A "nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof, combined 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 may be synthesized by any useful method, e.g., chemically, enzymatically, or recombinantly, and may contain one or more modified or non-natural nucleosides. A polynucleotide may contain one or more regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotide will contain regions of nucleotides.

[0200] Modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides (including 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. 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 may be incorporated into the polynucleotides of the present disclosure. Modifications of polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides) useful in the vaccines of the present disclosure 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-Methyladenosine;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,N 6-Dimethyladenosine;N6-Acetyladenosine;N6-Hydroxynorvalylcarbamoyladenosine;N6-Methyl-N6-threonylcarbamoyladenosine;2-Methyladenosine;2-Methylthio-N6-isopentenyladenosine;7-Deaza-adenosine;N1-Methyladenosine;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)adenosine 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'-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)adenine8(Thioalkyl)adenine;8-(Alkenyl)adenine;8-(Alkyl)adenine;8-(Alkynyl)adenine;8-(Amino)adenine;8-(Halo)adenine;8-(Hydroxyl)adenine;8-(Thioalkyl)adenine;8-(Thioyl)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-aminoadenosine 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'-b-aminoadenosine TP;2'-Deoxy-2'-b-azidoadenosine TP;2'-Deoxy-2'-b-bromoadenosine TP;2'-Deoxy-2'-b-chloroadenosine TP;2'-Deoxy-2'-b-fluoroadenosine TP;2'-Deoxy-2'-b-iodoadenosine TP;2'-Deoxy-2'-b-mercaptoadenosine TP;2'-Deoxy-2'-b-thiomethoxyadenosine TP;2-Fluoroadenosine TP;2-Iodoadenosine TP;2-Mercaptoadenosine ATP;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'-Ethynyladenosine TP;5'-Homoadenosine TP;8-Aza-ATP;8-Bromo-adenosine TP;8-Trifluoromethyladenosine 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-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;Lysine;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;3-(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; 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; 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'-a-mercapto Cytidine TP; 2'-deoxy-2'-a-thiomethoxycytidine TP; 2'-deoxy-2'-b-aminocytidine TP; 2'-deoxy-2'-b-azidocytidine TP; 2'-deoxy-2'-b-bromocytidine TP; 2'-deoxy-2'-b-chlorocytidine TP; 2'-deoxy-2'-b-fluorocytidine TP; 2'-deoxy-2'-b-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-Ethynylara-cytidine TP;5-Ethynylcytidine TP;5'-Homocytidine TP;5-Methoxycytidine TP;5-Trifluoromethyl-cytidine TP;N4-Aminocytidine TP;N4-Benzoyl-cytidine Din TP; Pseudoisocytidine; 7-Methylguanosine; N2,2'-O-Dimethylguanosine; N2-Methylguanosine; Wyosine; 1,2'-O-Dimethylguanosine; 1-Methylguanosine; 2'-O-Methylguanosine; 2'-O-Ribosylguanosine(phosphate); 2'-O-Methylguanosine; 2'-O-Ribosylguanosine(phosphate); 7-Aminomethyl-7-deazaguanosine; 7-Cyano-7-deazaguanosine; Archaeosine; Methylwyosine; 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)guanine;6-(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;8-(Halo)guanine;8-(Hydroxyl)guanine;8-(Thioalkyl)guanine;8-(Thioyl)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-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'-α-Ethynylguanosine TP;2'-α-Trifluoromethyl 2'-b-Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine T; P;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-Thio Uridine;3-Methyluridine;5-Carboxymethyluridine;5-Hydroxyuridine;5-Methyluridine;5-Taurinomethyl-2-thiouridine;5-Taurinomethyluridine;Dihydrouridine;Pseudouridine;(3-(3-amino-3-carboxypropyl)uridine;1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-Methylpseudouridine;1-Methyl-pseudouridine;2'-O-Methyluridine;2'-O-Methylpseudouridine;2'-O-Methyluridine;2-Thio-2'-O-methyl Uridine;3-(3-amino-3-carboxypropyl)uridine;3,2'-O-dimethyluridine;3-methyl-pseudo-uridine TP;4-thiouridine;5-(carboxyhydroxymethyl)uridine;5-(carboxyhydroxymethyl)uridine methyl ester;5,2'-O-dimethyluridine;5,6-dihydrouridine;5-aminomethyl-2-thiouridine;5-carbamoylmethyl-2'-O-methyluridine;5-carbamoylmethyluridine;5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyl Uridine methyl ester;5-carboxymethylaminomethyl-2'-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine;5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP;5-Methoxycarbonylmethyl-2'-O-methyluridine;5-Methoxycarbonylmethyl-2-thiouridine;5-Methoxycarbonylmethyluridine;5-Methoxyuridine;5-Methyl-2-thiouridine;5-Methylaminomethyl-2-selenouridine;5-Methylaminomethyl-2-thiouridine;5-Methylaminomethyluridine;5-Methyldihydrouridine;5-Hydroxyacetic acid-uridine TP;5-Hydroxyacetic acid-methyl ester-uridine TP;N1-Methyl-pseudouridine;N1-Ethyl-pseudouridine;Uridine 5-hydroxyacetic acid;Uridine 5-hydroxyacetic acid methyl ester;3-(3-amino-3-carboxypropyl)-uridine TP;5-(isopentenylaminomethyl)-2-thiouridine TP;5-(isopentenylaminomethyl)- 1-(aminoalkylaminomethyl)-2'-O-methyluridine TP;5-(iso-pentenylaminomethyl)uridine TP;5-propynyluracil;α-thiouridine;1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil;1-(aminoalkylaminocarbonylethylenyl)-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-(aminoalkylaminocarbonylethylenyl)-2-(thio)-pseudouracil;1-methyl-3-(3-amino-3-carboxypropyl)pseudouracil TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-methyl-pseudo-UTP; 2(thio)pseudouracil; 2'deoxyuridine; 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'-azidouridine TP;2-Methylpseudouridine;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(guanidiniumalkyl)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-(Alkyl)uracil;5-(Alkynyl)uracil;5-(Allylamino)uracil;5-(Cyanoalkyl)uracil;5-(Dialkylaminoalkyl)uracil;5-(Dimethylaminoalkyl)uracil;5-(Guanidiniumalkyl)uracil;5-(Halo)uracil;5-(1,3-Diazol-1-alkyl)uracil;5-(Methoxy)uracil;5-(Methoxycarbonylmethyl)-2-(thio)uracil;5-(Methoxycarbonylmethyl)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)uracil5-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-Carboxymethyl-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 (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 T P; 1-(2,2-diethoxyethyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethylphenyl)pseudouridine TP; 1-(2-amino-2-carboxyethyl)pseudouridine TP; 1-(2-aminoethyl)pseudouridine TP; 1-(2-hydroxyethyl)pseudouridine TP; 1-(2-methoxyethyl)pseudouridine TP; 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP; 1-(3-amino-3-carboxypropyl)pseudo-UTP; 1-(3-amino-propyl)pseudo-UTP; 1-(3-cyclopropyl-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)pseudo-UTP 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)pseudouridine TP;1-(4-methoxybenzyl)pseudouridine TP;1-(4-methoxyphenyl)pseudouridine TP;1-(4-methylbenzyl)pseudouridine TP;1-(4-methylbenzyl)pseudouridine TP;1-(4-nitrobenzyl )pseudouridine TP;1-(4-nitrobenzyl)pseudo-UTP;1(4-nitrophenyl)pseudo-UTP;1-(4-thiomethoxybenzyl)pseudouridine TP;1-(4-trifluoromethoxybenzyl)pseudouridine TP;1-(4-trifluoromethylbenzyl)pseudouridine TP;1-(5-aminopentyl)pseudo-UTP;1-(6-aminohexyl)pseudo-UTP;1,6-dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}- 1-(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-UTP; 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;1-cyclopentyl-pseudo-UTP;1-cyclopropylmethyl-pseudo-UTP;1-cyclopropyl-pseudo-UTP;1-Ethyl 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-ethylcaprylamide Carboxylate-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-Propargylpseudouridine TP;1-Propyl-pseudo-UTP;1-Propynyl-pseudouridine;1-p-Tolyl-pseudo-UTP;1-te rt-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'-b-Ethynyluridine TP;2'-b-Trifluoromethyluridine TP;2'-Deoxy-2',2'-difluorouridine TP;2'-Deoxy-2'-a-mercaptouridine TP;2'-Deoxy-2'-a-thiomethoxyuridine TP;2'-Deoxy-2'-b-aminouridine TP;2'-Deoxy-2'-b-azidouridine TP;2'-Deoxy-2'-b-bromouridine TP;2'-Deoxy-2'-b-chlorouridine TP;2'-Deoxy-2'-b-fluorouridine TP;2'-Deoxy-2'-b-iodo Uridine 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'-Homo-uridine TP;5-Iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-uridine 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; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP -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;6-isopropyl-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-methylbenzenesulfonic acid) TP;Pseudouridine 1-(4-methylbenzoic acid) TP;Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid;Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy) Pseudouridine TP1-[3-{2-(2-[2-(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-methylphosphonic acid;Pseudouridine TP1-methylphosphonic acid diethyl ester;Pseudo-UTP-N1-3-propionic acidPseudo-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; Unmodified hydroxywybutosin; 4-Demethylwybutosin; 2,6-(Diamino)purine; 1-(Aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-( Diaza)-2-(oxo)-phenthiazin-1-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 bases;2'-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl;2-oxo-pyridopyrimidin-3-yl;2-pyridinone;3-nitropyrrole;3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl;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)-phenthiazin-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-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aza)indolyl;7-(Guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3- (Aza)-phenthiazin-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)-phenoxazin-1-yl;7-(Propynyl)isocarbostyril 7-(Propynyl)isocarbostyrilyl, 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)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Diflu Olotryl;Hypoxanthine;Imidizopyridinyl;Inosinyl;Isocarbostyrilyl;Isoguanidine;N2-Substituted Purines;N6-Methyl-2-amino-purine;N6-Substituted Purines;N-Alkylated Derivatives;Naphthalenyl;Nitrobenzimidazolyl;Nitroimidazolyl;Nitroindalyl;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-amino-riboside-TP; formycin ATP; formycin BTP; pyrrolidine 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-pentaoxanonadecyl)adenosine TP.

[0201] In some embodiments, a polynucleotide (eg, an RNA polynucleotide, eg, an mRNA polynucleotide) comprises a combination of at least two (eg, 2, 3, 4 or more) of the above-described modified nucleobases.

[0202] In some embodiments, modified nucleobases in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) include pseudouridine (Ψ), N1-methylpseudouridine (m 1Ψ), 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-thiopseudouridine, 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, 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 above-described modified nucleobases.

[0203] In some embodiments, the modified nucleobase in a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) is 1-methyl-pseudouridine (m 1 Ψ), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine (Ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, the polynucleotide comprises a combination of at least two (e.g., 2, 3, 4, or more) of the above-described modified nucleobases.

[0204] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) contains pseudouridine (Ψ) and 5-methyl-cytidine (m 5 In some embodiments, the polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m 1 In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1 Ψ) and 5-methyl-cytidine (m 5 In some embodiments, the polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2-thiouridine (sC). 2 In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (mU). 5 In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises methoxy-uridine (moC). 5 In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 5-methoxy-uridine (moU). 5 U) and 5-methyl-cytidine (m 5 In some embodiments, the polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, the polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m 5 In some embodiments, the polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises an N6-methyl-adenosine (mC). 6 In some embodiments, the polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises an N6-methyl-adenosine (mA). 6 A) and 5-methyl-cytidine (m 5 C).

[0205] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) is uniformly modified (e.g., completely modified, modified throughout the entire sequence) for a particular modification. For example, the polynucleotide may be modified with 5-methyl-cytidine (m 5It may be uniformly modified with 5-methyl-cytidine (mC), which means that all cytosine residues in the mRNA sequence are modified with 5-methyl-cytidine (mC). 5 C). Similarly, polynucleotides may be uniformly modified by substituting any type of nucleoside residue present in the sequence with a modified residue as described above. 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.

[0206] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and in some embodiments, the modified nucleobase is a modified cytosine. Nucleosides having modified uridine include 5-cyanouridine and 4'-thiouridine. In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0207] In some embodiments, the modified nucleobase is 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, 7-methyl-8-oxo-guanosine.

[0208] The polynucleotides of the present disclosure may be partially or completely modified along the entire length of the molecule. For example, one or more, or all, or a predetermined type of nucleotide (e.g., purine or pyrimidine, or any one or all of A, G, U, C) may be uniformly modified in the polynucleotide of the present disclosure or in a predetermined predetermined sequence region thereof (e.g., in an mRNA including or excluding a polyA tail). In some embodiments, all nucleotides X in the polynucleotide of the present disclosure (or in a predetermined sequence region thereof) are modified nucleotides, where X may be 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.

[0209] Polynucleotides may contain from about 1% to about 100% modified nucleotides (with respect to overall nucleotide content, or with respect to any one or more types of nucleotides, i.e., A, G, U, or C) or any intervening percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% 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 is understood that the remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0210] A polynucleotide may contain from as little as 1% to as much as 100% modified nucleotides, or any intervening percentage, 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 may contain modified pyrimidines such as modified uracil or 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 may be substituted with modified uracils (e.g., 5-substituted uracils). The modified uracils may be replaced with a compound having a single unique structure, or with multiple compounds having 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 with a compound having a single unique structure, or may be replaced with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0211] In some embodiments, the codon-optimized RNA may have, for example, an enhanced G / C level. The G / C content of a nucleic acid molecule can affect the stability of the RNA. RNA with an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modification in the translated region. Due to the degeneracy of the genetic code, this modification works by replacing existing codons with those that promote greater RNA stability without changing the resulting amino acid. This approach is limited to the coding region of the RNA.

[0212] Thus, in some embodiments, the RNA (e.g., mRNA) vaccine comprises a 5' UTR element, optionally a codon-optimized open reading frame, and a 3' UTR element, a poly(A) sequence and / or a polyadenylation signal (the RNA is not chemically modified). In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (Ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine-5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-1-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester ( mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U ), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1Ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-di Hydrouridine, 5-methyl-dihydrouridine (m5D), 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, N1-ethyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3U), p3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine These include lysine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 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.

[0213] 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 (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-his(III)-cytidine (e.g., 5-iodo-cytidine), 5-hydroxy-cytidine (5-hydroxy ... Hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 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 cytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl These include 2'-F-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0214] 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, N6-methyl-adenine (m6A), 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (g6A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 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-pentaoxanonadecyl)-adenosine.

[0215] 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), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OhyW), and unmodified 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 (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2 ,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1 N-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0216] In vitro transcription of RNA (e.g., mRNA) The HCMV 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 from a template DNA, e.g., referred to as an "in vitro transcription template." In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The specific nucleic acid sequence composition and length of the in vitro transcription template depend on the mRNA encoded by the template.

[0217] "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 translated by the ribosome) that does not encode a polypeptide.

[0218] "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (i.e., 3') from a stop codon (i.e., the codon of the mRNA transcript that signals translation termination) that does not encode a polypeptide.

[0219] An "open reading frame" is a contiguous stretch of DNA beginning with a start codon (eg, methionine (ATG)) and ending with a stop codon (eg, TAA, TAG, or TGA) that encodes a polypeptide.

[0220] A "poly-A tail" is a region of an mRNA downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple consecutive adenosine monophosphates. The poly-A tail may contain 10 to 300 adenosine monophosphates. For example, the poly-A tail may 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, the poly-A tail contains 50 to 250 adenosine monophosphates. In relevant biological environments (e.g., intracellular, in vivo), poly(A) tails function to protect mRNA from enzymatic degradation (e.g., in the cytoplasm) and at transcription termination to assist in the transport and translation of mRNA from the nucleus.

[0221] In some embodiments, the polynucleotide comprises 200 to 3000 nucleotides. For example, the polynucleotide may comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0222] Treatment method Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of HCMV in humans and other mammals. HCMV RNA vaccines may be used as therapeutic or prophylactic agents. They may also be used in medicines for the prevention and / or treatment of infectious diseases. In exemplary embodiments, the HCMV RNA vaccines of the present invention are used to provide prophylactic protection from human cytomegalovirus infection and may be particularly useful in the prevention and / or treatment of immunocompromised and infant patients to prevent or reduce the severity and / or duration of clinical signs of cytomegalovirus infection. In some embodiments, the vaccines described herein reduce or prevent congenital transmission of HCMV from mother to child.

[0223] Broad-spectrum vaccines HCMV RNA (e.g., mRNA) vaccines may be used as therapeutic or prophylactic agents. It is anticipated that situations may exist in which a person is at risk of infection with more than one betacoronavirus (e.g., at risk of HCMV infection). RNA (e.g., mRNA) therapeutic vaccines are particularly suited to combination vaccination approaches due to numerous factors, including, but not limited to, speed of production and the ability to rapidly adjust vaccines to adapt to perceived geographic threats. Furthermore, because vaccines utilize the human body to produce antigenic proteins, they are amenable to the production of larger, more complex antigenic proteins, allowing for proper folding, surface expression, antigen presentation, etc., in human subjects. To protect against more than one HCMV strain, a combination vaccine may be administered that contains RNA encoding at least one antigenic polypeptide of a first HCMV and also contains RNA encoding at least one antigenic polypeptide of a second HCMV. The RNAs (mRNAs) may be co-formulated, for example, in a single LNP or in separate LNPs intended for simultaneous administration.

[0224] A method for inducing an immune response against HCMV in a subject is provided as an embodiment of the present invention. The method comprises administering to the subject an HCMV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the HCMV antigen polypeptide or immunogenic fragment thereof, wherein the anti-antigen polypeptide antibody titer in the subject is increased after vaccination relative to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV. An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigen polypeptide.

[0225] A prophylactically effective dose is a therapeutically effective dose that prevents infection by a virus at a clinically acceptable level. In some embodiments, a therapeutically effective dose is a dose listed in the package insert of the vaccine. As used herein, a traditional vaccine refers to a vaccine other than the mRNA vaccine of the present invention. For example, traditional vaccines include, but are not limited to, live microbial vaccines, killed microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, etc. In an exemplary embodiment, a traditional vaccine is one that has regulatory approval and / or is registered with a national drug regulatory agency, e.g., the Food and Drug Administration (FDA) or the European Medicines Agency (EMA) in the United States.

[0226] In some embodiments, the anti-antigen polypeptide antibody titer in the subject is increased by 1 log to 10 logs after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV.

[0227] In some embodiments, the anti-antigen polypeptide antibody titer in the subject is increased by one log after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV.

[0228] In some embodiments, the anti-antigen polypeptide antibody titer in the subject increases by 2 logs after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV. In some embodiments, the anti-antigen polypeptide antibody titer in the subject increases by 3 logs after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV.

[0229] In some embodiments, the anti-antigen polypeptide antibody titer in the subject is increased by 5 logs after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV.

[0230] In some embodiments, there is a 10 log increase in anti-antigen polypeptide antibody titers following vaccination compared to subjects vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV. Another embodiment of the present invention provides a method for inducing an immune response against HCMV in a subject, comprising administering to the subject an HCMV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one HCMV antigen polypeptide or immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the HCMV antigen polypeptide or immunogenic fragment thereof, wherein the immune response in the subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine against HCMV at a dose level 2-100 times higher than that of the RNA vaccine.

[0231] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at twice the dose level compared to an HCMV RNA vaccine.

[0232] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at a dose level three times higher compared to an HCMV RNA vaccine. In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at four times the dose level compared to an HCMV RNA vaccine.

[0233] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject who receives a traditional vaccination at a 5-fold dose level compared to an HCMV RNA vaccine.

[0234] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at a 10-fold higher dose level compared to an HCMV RNA vaccine.

[0235] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at a 50-fold higher dose level compared to an HCMV RNA vaccine.

[0236] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at a 100-fold dose level compared to an HCMV RNA vaccine.

[0237] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at a dose level 10-1000 times higher than that of an HCMV RNA vaccine.

[0238] In some embodiments, the immune response in a subject is equivalent to the immune response in a subject vaccinated with a traditional vaccine at a dosage level 100-1000 times higher than that of an HCMV RNA vaccine.

[0239] In other embodiments, the immune response is assessed by measuring anti-antigen polypeptide antibody titers in the subject.

[0240] In another aspect, the present invention provides a method for eliciting an immune response in a subject against HCMV by administering to the subject an HCMV RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one HCMV antigenic polypeptide or immunogenic fragment thereof, thereby eliciting an immune response in the subject specific for the HCMV antigenic polypeptide or immunogenic fragment thereof, wherein the immune response in the subject is induced 2 to 10 weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine against HCMV. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine at a dose 2- to 100-fold higher than that of the RNA vaccine.

[0241] In some embodiments, an immune response in a subject is induced two days earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0242] In some embodiments, an immune response in a subject is induced three days earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0243] In some embodiments, an immune response in a subject is induced one week earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0244] In some embodiments, an immune response in a subject is induced two weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0245] In some embodiments, an immune response in a subject is induced three weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0246] In some embodiments, an immune response in a subject is induced five weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0247] In some embodiments, an immune response in a subject is induced 10 weeks earlier than the immune response induced in a subject vaccinated with a prophylactically effective amount of a traditional vaccine.

[0248] Also provided herein is a method for eliciting an immune response in a subject against HCMV by administering to the subject an HCMV RNA vaccine having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not contain a stabilizing element and an adjuvant is not co-formulated or co-administered with the vaccine.

[0249] Standards of Care for CMV Prevention and Treatment Various approaches to prevent and / or treat CMV have been previously pursued or are currently underway, including immunization strategies, some of which are summarized below. However, all of these approaches have drawbacks and limitations. (Schleiss et al. (2008), Curr Top Microbiol Immunol. 325:361-382).

[0250] Ganciclovir and valganciclovir In some embodiments, ganciclovir or valganciclovir are standard of care regimens for the treatment or prevention of CMV infection (Reusser P. et al. (2000); 130(4):101-12; Biron et al. (2006) Antiviral Research 71:154-163).

[0251] Ganciclovir (sold as CYTOVENE® and ZIRGAN®) and valganciclovir (a prodrug form of ganciclovir marketed as VALCYTE®) are antiviral drugs developed by Hoffmann-La Roche to treat CMV infection. They are analogs of 2'-deoxy-guanosine, which competitively inhibit the incorporation of dGTP into DNA and subsequent viral replication (Sugawara M et al., J Pharm Sci. 2000;89(6):781-9). CYTOVENE-IV (ganciclovir sodium for injection) is FDA-approved "only for use in the treatment of cytomegalovirus (CMV) retinitis in immunocompromised patients and for the prevention of CMV disease in transplant patients at risk for CMV disease" (FDA Label, 1 / 31 / 2006, page 1).

[0252] The recommended dosing regimen for CYTOVENE-IV for the treatment of CMV retinitis in patients with normal renal function includes an induction phase of 5 mg / kg (administered intravenously over 1 hour) every 12 hours for 14 to 21 days, followed by a maintenance phase of 5 mg / kg (administered intravenously over 1 hour) once daily, 7 days a week, or 6 mg / kg once daily, 5 days a week. (Id., p. 22) For CMV prophylaxis in transplant patients with normal renal function, the recommended dosing regimen includes 5 mg / kg (administered intravenously over 1 hour) every 12 hours for 7 to 14 days, followed by 5 mg / kg once daily, 7 days a week, or 6 mg / kg once daily, 5 days a week. (Id.)

[0253] In a study of heart transplant patients 120 days after transplant, the incidence of CMV in seropositive subjects was 9% in treated subjects compared with 46% in subjects given a placebo. (Biron et al. (2006) Antiviral Research 71:154-163, page 157). In a study of bone marrow transplant subjects, the incidence of CMV in treated subjects was 3% compared with 43% in placebo-treated subjects 100 days after transplant. (Ibid.). One form of ganciclovir, sold by Bausch and Lomb (ZIRGAN®), is in the form of an ophthalmic gel approved by the FDA for the treatment of acute herpetic keratitis (dendritic ulcers) (FDA label, 9 / 15 / 2009, page 4; Wilhelmus KR et al., 2010, Cochrane Database Syst Rev 12:CD002898).

[0254] VALCYTE® (valganciclovir hydrochloride) in tablet form is FDA-approved in adult patients for the treatment of CMV retinitis in patients with acquired immunodeficiency syndrome (AIDS) and for the prevention of CMV disease in high-risk kidney, heart, and kidney-pancreas transplant recipients. (FDA label, April 23, 2015, page 1) The dosing regimen for VALCYTE®, as shown in the FDA label dated April 23, 2015, is shown in the table below. [Table 1]

[0255] Oral forms of ganciclovir have been found to have low bioavailability (Biron et al. (2006) Antiviral Research 71:154-163). Valganciclovir has been reported to have better bioavailability than ganciclovir (Pescovitz MD et al., Antimicrob Agents Chemother. 2000;44(10):2811-5; Biron et al. (2006) Antiviral Research 71:154-163).

[0256] Adverse side effects associated with ganciclovir and valganciclovir include fever, rash, diarrhea, and hematologic effects (such as neutropenia, anemia, and thrombocytopenia), as well as potential reproductive toxicity. Ganciclovir has also been shown to affect fertility in animal studies and to have carcinogenic and teratogenic effects. (Biron et al. (2006) Antiviral Research 71:154-163)

[0257] Phase 3 clinical trials involving treatment of CMV infection with ganciclovir or valganciclovir include trials associated with the following clinical trial government identification numbers: NCT00000143, NCT00000136, NCT00000134, NCT00497796, NCT00227370, NCT00466817, and NCT00294515. Results of clinical trials involving ganciclovir or valganciclovir are summarized in Biron et al. (2006) Antiviral Research 71:154-163, which is incorporated herein by reference in its entirety.

[0258] Experimental vaccines in development for CMV TransVax™ (also known as ASP0113 and VCL-CB01) TransVax™ is a CMV vaccine being developed by Vical Incorporated and Astellas Pharma Inc. (Smith et al. (2013) Vaccines 1(4):398-414). TransVax™ is a bivalent DNA vaccine containing a plasmid encoding CMV pp65 and gB antigens formulated in CRL1005 poloxamer and benzalkonium. (Ibid.; Kharfan-Dabaja et al. (2012) Lancet Infect Dis 12:290-99). The pp65 antigen induces a cytotoxic T cell response and confers cellular immunity, while the gB antigen induces both cellular immunity and antigen-specific antibody production. Thus, the vaccine is intended to induce both cellular and humoral immune responses. The pp65 and gB sequences have been modified from the wild-type protein sequences by deletion and codon optimization as described in Smith et al. (2013) Vaccines 1(4):398-414, pages 402-403, which is incorporated herein by reference in its entirety.

[0259] TransVax™ has received orphan drug designation in the US and Europe for hematopoietic stem cell transplantation (HSCT), including bone marrow transplant and solid organ transplant (SOT) patients.

[0260] In a phase 1 clinical trial, 37.5% and 50% of CMV subjects receiving 1 mg and 5 mg doses of the vaccine, respectively, demonstrated antibody or T-cell responses (Smith et al. (2013) Vaccines 1(4):398-414, p. 406). A phase 2 clinical trial (ClinicalTrials.gov identification number NCT00285259) was conducted in patients undergoing allogeneic hematopoietic stem cell transplantation (Kharfan-Dabaja et al. (2012) Lancet Infect Dis 12:290-99). Transplant patients received four doses of the experimental vaccine, including one pre-transplant dose (ibid., p. 292). The pre-transplant dose was administered 3-5 days before transplantation, while post-transplant doses were administered 21-42 days and 84 and 196 days after transplantation (ibid.). Endpoints included evaluation of safety and reduction in cytomegalovirus viremia (ibid.). The incidence of cytomegalovirus viremia was found to be lower in patients who received the vaccine compared with placebo (32.5% (vaccine group) compared with 61.8% (placebo); Table 2, p. 294, of Kharfan-Dabaja et al.). The vaccine has also been reported to be well tolerated and safe (ibid., p. 295). However, the incidence of viremia requiring antiviral treatment after vaccine treatment was similar to that of placebo controls (ibid., p. 296).

[0261] TransVax™ is currently being tested in a Phase 3 clinical trial for the treatment of hematopoietic cell transplant (HCT) patients under ClinicalTrials.gov identification number NCT01877655. The study endpoints are mortality and peripheral organ disease (EOD) in the first year after transplant. Estimated enrollment is 500, and the vaccine is administered by intramuscular injection. TransVax™ is also currently being tested in a Phase 2 clinical trial in CMV-seronegative kidney transplant recipients receiving organs from CMV-seropositive donors under ClinicalTrials.gov identification number NCT01974206. The primary outcome measured in this trial is the incidence of CMV viremia one year after the first dose of the drug. Enrollment is 150, and the vaccine is administered by intramuscular injection. Subjects enrolled in the trial were randomized to receive either ganciclovir or valganciclovir within 10 days of transplant.

[0262] Clinical trials involving TransVax™ can be found on the ClinicalTrials.gov website under the following ClinicalTrials.gov identifiers: NCT02103426, NCT01877655, NCT01974206, and NCT01903928.

[0263] U.S. patents and published applications assigned to Vical Inc. and related to CMV include: U.S. Patent Nos. 8,673,317, 9,180,162, 8,278,093, 7,888,112, and 7,410,795, which are incorporated by reference herein in their entireties.

[0264] An experimental vaccine under development by City of Hope / National Cancer Institute / Helocyte Several experimental CMV vaccines are under development by City of Hope and its licensee, Helocyte. U.S. patents and published applications assigned to City of Hope that relate to CMV include: U.S. Patent Nos. 7,387,782, 7,025,969, 6,133,433, 6,207,161, 6,074,645, 6,251,399, 6,727,093, 6,726,910, 6,843,992, and 6 ,544,521, 6,951,651, 8,580,276, 7,163,685, 6,242,567, 6,835,383, 6,156,317, 6,562,345, U.S. Patent Application Publication Nos. 2014-0065181 and 2015-0216965 (incorporated herein by reference in their entireties).

[0265] CMVPepVax CMVPepVax is an experimental vaccine being developed by City of Hope Medical Center, the National Cancer Institute, and Helocyte, Inc. This vaccine contains a pp65 T cell epitope and a tetanus T helper epitope in the form of a chimeric peptide, and also contains the adjuvant PF03512676 (Nakamura R et al., Lancet Heamatology (2016) Feb;3(2):e87-98). CMVPepVax was tested in a phase 1b clinical trial in CMV-seropositive patients undergoing hematopoietic stem cell transplantation (Ibid.). The vaccine was administered subcutaneously on days 28 and 56 (Ibid.). Patients who received the vaccine were reported to have improved recurrence-free survival (Ibid.). This clinical trial has been assigned the ClinicalTrials.gov identification number NCT01588015. CMVPepVax is currently in a phase 2 clinical trial under ClinicalTrials.gov identification number NCT02396134 to determine its effectiveness in reducing the frequency of cytomegalovirus events in patients with hematologic malignancies who have undergone donor stem cell transplantation.

[0266] CMV-MVA triplex CMV-MVA-triplex is an experimental CMV vaccine being developed by City of Hope Medical Center, the National Cancer Institute, and Helocyte, Inc. (formerly DiaVax Biosciences). The vaccine consists of an inactivated modified vaccinia Ankara (MVA) virus vector encoding the CMV antigens UL83 (pp65), UL123 (IE1), and UL122 (IE2). (NCI Drug Dictionary)

[0267] The CMV-MVA triplex is currently being tested in a Phase 2 clinical trial to assess its efficacy in reducing CMV complications in patients previously infected with CMV and undergoing donor hematopoietic cell transplantation. This trial has been assigned ClinicalTrials.gov identification number NCT02506933. A Phase 1 clinical trial in healthy volunteers with or without previous CMV exposure is also underway (ClinicalTrials.gov identification number NCT01941056).

[0268] pentamer City of Hope and Helocyte, Inc. are also pursuing a pentameric vaccine using a modified vaccinia Ankara (MVA) virus vector encoding five CMV pentameric subunits. This vaccine is still in preclinical development. (Wussow et al. (2014) PLoS Pathog 10(11):e1004524. doi:10.1371 / journal.ppat.1004524)

[0269] gB / MF59 This experimental vaccine, originally developed in the 1990s, combines the gB antigen with the MF59 adjuvant (Pass et al. (2009) J Clin Virol 46(Suppl 4):S73-S76). Several clinical trials sponsored by Chiron Corporation in the 1990s showed that the vaccine was safe (ibid., p. 2). Sanofi Pasteur later acquired the rights to the vaccine (ibid.).

[0270] A phase 2 clinical trial was conducted in postpartum women beginning in 1999 (enrollment completed in 2006) with an endpoint of CMV infection (ibid., p. 3). Subjects were administered the vaccine at 0, 1, and 6 months. (Rieder et al. (2014) Clin Microbiol Infect 20(Suppl. 5):95-102, p. 98). CMV infection was diagnosed in 8% of vaccine-treated subjects compared with 14% of placebo-treated subjects (corresponding to an efficacy of 43%, respectively). Results showed a 50% reduction in CMV infection rates in vaccine-treated subjects (3.3% in test subjects compared with 6.6% in placebo-treated subjects) (ibid.; Pass et al. (2009) J Clin Virol 46(Suppl. 4):S73-S76, p. 4). A 50% reduction in CMV infection rates has been described as "lower than desired from a clinical perspective" (Rieder et al. (2014) Clin Microbiol Infect 20(Suppl. 5):95-102, page 98).

[0271] Phase 2 clinical trials are also being conducted with gB / MF59 in kidney and liver transplant patients (ibid., p. 100). It was reported that "high gB antibody titers correlated with a shorter duration of viremia" and that "the duration of viremia and the number of days of ganciclovir treatment were reduced" (ibid.).

[0272] Clinical trials targeting gB / MF59 can be found on the ClinicalTrials.gov website under the following ClinicalTrials.gov identifiers: NCT00133497, NCT00815165, and NCT00125502.

[0273] U.S. Patent Application No. 2009-0104227, assigned to Sanofi Pasteur SA, is incorporated herein by reference in its entirety.

[0274] gB / AS01 GlaxoSmithKline is developing an experimental vaccine containing the gB antigen combined with the AS01 adjuvant. (McVoy (2013) Clinical Infectious Diseases 57(S4):S196-9, page S197) This vaccine is called GSK1492903A. Clinical trials for GSK1492903A can be found on the ClinicalTrials.gov website under the following ClinicalTrials.gov identifiers: NCT00435396 and NCT01357915.

[0275] WO2016 / 067239 and WO2015 / 181142 filed by GlaxoSmithKline Biologicals SA are incorporated herein by reference in their entireties.

[0276] Towne Vaccine The Towne vaccine for CMV is a live attenuated vaccine. (McVoy (2013) Clinical Infectious Diseases 57(S4):S196-9, page S197) This vaccine, at least when administered at low doses, was unsuccessful in protecting against primary maternal infection. (ibid.) In a study of kidney transplant recipients, treatment with this vaccine resulted in a reduction in severe disease while minimizing the impact on mild disease. (Plotkin et al. (1994) Transplantation 58(11):1176-8)

[0277] Live attenuated vaccines have also been developed in which segments of the Towne genome have been replaced with sequences from other "low passage" strains; these have been termed "Towne-Toledo chimeras" and have been found to be well tolerated in Phase 1 clinical trials (McVoy (2013) Clinical Infectious Diseases 57(S4):S196-9, page S197; Heineman et al. (2006) The Journal of Infectious Diseases 193:1350-60). Chimeric viral genomes containing portions of the Towne genome are described in U.S. Patent No. 7,204,990, which is incorporated herein by reference in its entirety.

[0278] Another approach being investigated involves co-administering the Towne vaccine with the adjuvant recombinant interleukin-12 (rhIL-12) (Jacobson et al. (2006) Vaccine 24:5311-9).

[0279] CMV-CTL The CMV-targeted T cell program (CMV-CTL) is a cellular immunotherapy approach being developed by Atara Biotherapeutics.

[0280] In a phase 1 clinical trial, monocytes were pulsed with CMV pp65 or a pp65 / IE1 peptide mixture to expand CMV CTLs and examine their immunological effects (Bao et al. (2012) J Immunother 35(3):293-298). CMV-specific immune responses were observed in approximately 70% of subjects who received CTLs (ibid., p. 5).

[0281] A phase 2 clinical trial is currently underway investigating third-party donor-derived CMVpp65-specific T cells for the treatment of CMV infection or persistent CMV viremia after allogeneic hematopoietic stem cell transplantation. This trial has been assigned ClinicalTrials.gov identification number NCT02136797. A phase 2 clinical trial is also underway investigating primary transplant donor-derived CMVpp65-specific T cells for the treatment of CMV infection or persistent CMV viremia after allogeneic hematopoietic stem cell transplantation. This trial has been assigned ClinicalTrials.gov identification number NCT01646645.

[0282] Monoclonal Ab Novartis CSJ148, being developed by Novartis, is a combination of two monoclonal antibodies targeting gB and the CMV pentameric complex (Dole et al. (2016) Antimicrob Agents Chemother. Apr 22;60(5):2881-7). The two antibodies are known as LJP538 and LJP539 (ibid.). LJP538, LJP539, and CSJ148 were found to be safe when administered intravenously to healthy volunteers and demonstrated predictable IgG pharmacokinetics (ibid.). CSJ148 is currently undergoing a phase 2 clinical trial investigating its efficacy and safety in stem cell transplant patients (ClinicalTrials.gov identification number NCT02268526).

[0283] Theraclone TCN-202 is a fully human monoclonal antibody being developed by Theraclone for the treatment of CMV infection. TCN-202 was found to be safe and well-tolerated in a Phase 1 clinical trial (ClinicalTrial.gov identification number NCT01594437). A Phase 2 trial began in 2013 to investigate efficacy in kidney transplant recipients. (Theraclone press release, September 10, 2013).

[0284] Brincidofovir Brincidofovir (CMX001) is an experimental lipid-nucleotide conjugate being developed by Chimerix, Durham, NC, for the treatment of DNA viruses, including CMV. Brincidofovir has received fast track designation from the FDA for CMV.

[0285] The results of a Phase 3 clinical trial (called "SUPPRESS") investigating CMV prevention in subjects undergoing hematopoietic cell transplantation (HCT) were announced in February 2016 (Chimerix Press Release, February 20, 2016). While the trial did not meet its primary endpoint of preventing CMV at 24 weeks, it reported that antiviral activity was observed during the treatment phase (ibid.). The trial involved 452 subjects undergoing HCT who received brincidofovir twice weekly for up to 14 weeks (ibid.). It was speculated that increased use of immunosteroids, such as corticosteroids for the treatment of graft-versus-host disease (GVHD), after brincidofovir treatment may have contributed to the failure to reach the primary endpoint (ibid.). While other Phase 3 trials were terminated based on the results of the SUPPRESS trial, Chimerix indicated its intention to further advance a Phase 2 trial in subjects undergoing kidney transplantation (ibid.). Information about clinical trials related to brincidofovir can be found on the ClinicalTrials.gov website, including the following identifiers: NCT02087306, NCT02271347, NCT02167685, NCT02596997, NCT02439970, NCT00793598, NCT01769170, NCT00780182, NCT01241344, NCT00942305, NCT02420080, NCT02439957, NCT01143181, and NCT01610765.

[0286] V160 V160 is an experimental CMV vaccine being developed by Merck based on the attenuated AD169 strain. V160 is currently being tested in a Phase 1 clinical trial evaluating a three-dose regimen testing multiple formulations in healthy adults. This trial has been assigned ClinicalTrials.gov identification number NCT01986010.

[0287] Merck is also pursuing a vaccine targeting the CMV pentameric complex (Loughney et al. (2015) jbc.M115.652230). U.S. patents and published applications assigned to Merck Sharp & Dohme Corp include U.S. Patent No. 2014-0220062 and U.S. Patent No. 2015-0307850, which are incorporated by reference in their entireties.

[0288] Letermovil Letermovir (AIC246) is an antiviral drug being developed by Merck for the treatment of CMV infection (Chemaly et al. (2014) New England Journal of Medicine, 370;19, May 8, 2014, Verghese et al. (2013) Drugs Future May;38(5):291-298). Letermovir was tested in a Phase IIb clinical trial for the prevention of CMV in HSCT recipients, ClinicalTrials.gov identification number NCT01063829, and was found to reduce the incidence of CMV infection in transplant recipients.

[0289] Redvax GmbH / Pfizer A preclinical candidate targeting CMV was developed by Redvax GmbH, a spin-off from Redbiotec AG, and is currently being pursued by Pfizer.

[0290] Patents and patent publications assigned to Redvax GmbH or Pfizer that relate to CMV include: U.S. Patent Application Publication Nos. 2015-0322115, WO2015 / 170287, U.S. Patent Application Publication Nos. 2015-0359879 and WO2014 / 068001, which are incorporated by reference in their entireties.

[0291] Therapeutic and Prophylactic Compositions Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, or diagnosis of HCMV in humans. HCMV RNA vaccines can be used as therapeutic or prophylactic agents. They can be used in medicine to prevent and / or treat infectious diseases. In some embodiments, the HCMV vaccines of the present invention can be used to prime immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, and then infused (reinfused) into a subject. In exemplary embodiments, an HCMV vaccine containing an RNA polynucleotide described herein may 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. In some embodiments, the subject is a woman of childbearing age. In some embodiments, the vaccine described herein reduces or prevents congenital transmission of HCMV from mother to child (Pass et al. (2014) J Ped Infect Dis 3(suppl 1):S2-S6).

[0292] An HCMV 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 envisioned. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of a composition comprising an HCMV RNA vaccine that includes a polynucleotide having at least one translatable region encoding an antigenic polypeptide. An "effective amount" of an HCMV RNA vaccine is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size and extent of modified nucleosides) and other components of the HCMV RNA vaccine, and other determinants. Generally, an effective amount of an HCMV RNA vaccine composition will elicit or enhance an immune response, preferably more efficiently as a function of intracellular antigen production, than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production can be demonstrated by increased cell transfection (percentage of cells transfected with the RNA vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., increased duration of protein translation from a modified polynucleotide), or a change in the antigen-specific immune response of host cells.

[0293] In some embodiments, an RNA vaccine according to the present disclosure (including a polypeptide encoding a polynucleotide) may be used to treat HCMV. The HCMV RNA vaccine may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals, or may be administered early in the incubation phase of infection or during active infection after symptom onset. In some embodiments, the amount of the RNA vaccine of the present disclosure provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

[0294] The HCMV RNA vaccine may be administered together with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or booster. As used herein, when referring to a prophylactic composition such as a vaccine, the term "booster immunization" refers to an extra administration of a prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The administration time between the initial administration of the prophylactic composition and the booster administration can 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, 4 The administration period may be greater than 1 day, 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 99 years. In exemplary embodiments, the administration period between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0295] In some embodiments, HCMV RNA vaccines can be administered intramuscularly or intradermally, similar to the administration of inactivated vaccines known in the art. HCMV RNA vaccines can be used in a variety of situations, depending on the prevalence of infection or the degree or level of unmet medical need. As a non-limiting example, RNA vaccines can be used to treat and / or prevent various infectious diseases. RNA vaccines have the advantage of generating greater antibody titers and generating responses more quickly than commercially available antiviral agents.

[0296] Provided herein are pharmaceutical compositions comprising HCMV RNA vaccines and RNA vaccine compositions and / or complexes, optionally in combination with one or more pharmaceutically acceptable excipients.

[0297] The HCMV RNA vaccine may be formulated or administered alone or in combination with one or more other components. For example, the HCMV RNA vaccine (vaccine composition) may contain other components, including, but not limited to, an adjuvant. In some embodiments, the HCMV RNA vaccine does not contain an adjuvant (they are adjuvant-free).

[0298] The HCMV RNA vaccine may be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the vaccine composition includes at least one additional active agent, such as a therapeutically effective agent, a prophylactically effective agent, or a combination of both. The vaccine composition may be sterile, pyrogen-free, or 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).

[0299] In some embodiments, the HCMV RNA vaccine is administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "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. Formulations of the vaccine compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such methods of preparation involve bringing into association the active ingredient (e.g., mRNA polynucleotide) with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, forming, and / or packaging the product into the desired single- or multi-dose units.

[0300] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will 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. By way of example, the composition may comprise 0.1-100%, e.g., 0.5-50%, 1-30%, 5-80%, or at least 80% (w / w) active ingredient.

[0301] HCMV RNA vaccines may be formulated with one or more excipients to (1) increase stability, (2) increase cell transfection, (3) enable sustained or delayed release (e.g., from depot formulations), (4) alter biodistribution (e.g., target specific tissues or cell types), (5) increase translation of the encoded protein in vivo, and / or (6) modify the release profile of the encoded protein (antigen) in vivo. In addition to any and all traditional excipients, such as solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickeners or emulsifiers, preservatives, excipients may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the HCMV RNA vaccine (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0302] Stabilizing Element Naturally occurring eukaryotic mRNA molecules have been found to contain stabilizing elements, including but not limited to untranslated regions (UTRs), at their 5'-end (5'UTR) and / or 3'-end (3'UTR), 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 elements of early mRNAs. Characteristic structural features of mature mRNAs, such as the 5'-cap and 3'-poly(A) tail, are usually added to transcribed (early) mRNAs during mRNA processing. The 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3' end of the transcribed mRNA. It may 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 individual mRNAs.

[0303] In some embodiments, the RNA vaccine may contain one or more stabilizing elements. Examples of stabilizing elements include histone stem loops. A 32-kDa protein, stem-loop binding protein (SLBP), has been identified. It 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; it peaks during S phase, when histone mRNA levels also increase. This protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by U7 snRNP. SLBP remains associated with the stem loop after processing and then stimulates translation of mature histone mRNA into histone protein 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 structure of the loop. The minimal binding site contains at least three nucleotides 5' and two nucleotides 3' to the stem loop. 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 should generally enhance 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)).

[0304] In some embodiments, the combination of a poly(A) sequence or polyadenylation signal with at least one histone stem loop acts synergistically to increase protein expression above levels observed with either of the individual elements, even though the two essentially represent alternative mechanisms. The synergistic effect of the combination of poly(A) with 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.

[0305] 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 approximately 15-20 nucleotides 3' of a naturally occurring stem-loop that represents a binding site for U7 snRNA, which is involved in processing histone pre-mRNA into mature histone mRNA. Ideally, the nucleic acids of the invention do not contain introns.

[0306] 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 generally involve intramolecular base pairing of two adjacent, partially or completely reverse-complementary sequences separated by a spacer, consisting of a short sequence derived from a histone gene that forms a structural loop. The unpaired loop region typically cannot base pair with either stem-loop element. This occurs more frequently in RNA because it is an important component of many RNA secondary structures, but can also occur in single-stranded DNA. The stability of the stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may occur. In some embodiments, at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0307] In other embodiments, RNA vaccines may have one or more AU-rich sequences removed. These sequences, sometimes called AURES, are destabilizing sequences found in the 3'UTR. AURES may be removed from RNA vaccines. Alternatively, AURES may remain in RNA vaccines.

[0308] Nanoparticle formulations In some embodiments, the HCMV RNA vaccine is formulated in nanoparticles. In some embodiments, the HCMV RNA vaccine is formulated in lipid nanoparticles. In some embodiments, the HCMV RNA vaccine is formulated in lipid-polycation complexes called cationic lipid nanoparticles. Formation of lipid nanoparticles may be achieved by methods known in the art and / or as described in U.S. Patent Application Publication No. 20120178702, the entire contents of which are incorporated herein by reference. Non-limiting examples of polycations include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine, and cationic peptides described in International Application No. WO2012013326 or U.S. Patent Application Publication No. US20130142818, each of which is incorporated herein by reference in its entirety. In some embodiments, the HCMV RNA vaccine is formulated in lipid nanoparticles containing non-cationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0309] Lipid nanoparticle formulations can be affected by, but are not limited to, the selection of cationic lipid components, cationic lipid saturation, the nature of PEGylation, the ratio of all components and biological parameters, such as size.In one example by Semple et al. (Nature Biotech, 2010 28:172-176; its entirety is incorporated herein by reference), lipid nanoparticle formulations are composed of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol and 1.4% PEG-c-DMA.In another example, by changing the composition of cationic lipids, siRNA can be more effectively delivered to various antigen-presenting cells (Basha et al., Mol Ther. 2011 19:2186-2200; its entirety is incorporated herein by reference).

[0310] 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.

[0311] In some embodiments, the ratio of PEG in a lipid nanoparticle formulation may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified 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 comprise 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% PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]]-1,2-dimyristyloxypropyl-3-amine (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, the PEG-c-DOMG is PEG Lipids may be substituted, for example, 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). Cationic lipids 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.

[0312] In some embodiments, the HCMV RNA vaccine formulation is a nanoparticle comprising at least one lipid. The lipid 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, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipid may be a lipid described in and / or produced by the method described in U.S. Patent Application Publication No. US20130150625, the entire contents of which are incorporated herein by reference. 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 in U.S. Patent Application Publication No. 20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 in U.S. Patent Application Publication No. 20130150625); [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 of U.S. Patent Application Publication No. 20130150625); 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 U.S. Patent Application Publication No. 20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0313] Lipid nanoparticle formulations typically contain lipids, particularly ionizable 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), as well as neutral lipids, sterols, and molecules that can reduce particle aggregation, such as PEG or PEG-modified lipids.

[0314] 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; and 0.5-15% PEG-lipid.

[0315] In some embodiments, the lipid nanoparticle formulation comprises 25% to 75% by molar of a cationic lipid selected from 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), e.g., 35% to 65%, 45% to 65%, 60%, 57.5%, 50%, or 40% by molar.

[0316] In some embodiments, the lipid nanoparticle formulation comprises 0.5-15% on a molar basis, e.g., 3-12%, 5-10%, 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%-50% on a molar basis of a sterol (e.g., 15-45%, 20-40%, 40%, 38.5%, 35%, or 31% on a molar basis). A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5%-20% on a molar basis of PEG or PEG-modified lipid (e.g., 0.5-10%, 0.5-5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis). In some embodiments, the PEG or PEG-modified lipid is 0.5%-20% on a molar basis. The modified lipid comprises PEG molecules with an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2,000, for example, 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), PEG-cDMA (further discussed in Reyes et al., J. Controlled Release, 107, 276-287 (2005) the contents of which are incorporated herein by reference in their entirety).

[0317] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 25-75% cationic lipid selected from 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 or PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 35-65% cationic lipid selected from 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 or PEG-modified lipid.

[0318] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 45-65% cationic lipid selected from 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 or PEG-modified lipid.

[0319] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 60% cationic lipid selected from 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 or PEG-modified lipid.

[0320] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid selected from 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 or PEG-modified lipid.

[0321] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid selected from 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 or PEG-modified lipid, and 0.5% targeting lipid.

[0322] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 40% cationic lipid selected from 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 or PEG-modified lipid.

[0323] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.2% cationic lipid selected from 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 or PEG-modified lipid.

[0324] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.5% cationic lipid selected from 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 or PEG-modified lipid.

[0325] 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.

[0326] In some embodiments, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol % of cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1134.3 / 1.4 (mol % of cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol % of cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0. ... 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 % of cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol % of cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), or 52 / 13 / 30 / 5 (mol % of cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

[0327] Non-limiting examples of lipid nanoparticle compositions and methods for their production 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. In some embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a PEG lipid, and a structured lipid, and may optionally contain a non-cationic lipid. As a non-limiting example, the lipid nanoparticle 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 nanoparticle 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 nanoparticle 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.

[0328] 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.

[0329] In some embodiments, the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-KC2-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DMG, and 38.5% structured 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.

[0330] In some embodiments, the nanoparticles comprise a compound of formula (I): [ka] or a salt or isomer thereof, wherein: R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR", -YR", and -R"M'R'; R2 and R3 are H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR”, or R2 and R3, which together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)N(R), -C(O)N(R)OR, and -C(R)N(R)C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, and aryl and heteroaryl groups; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * selected from the group consisting of Y-R", -Y-R", and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * is independently C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0331] In some embodiments, a subset of compounds of formula (I) is the following, wherein R4 is -(CH2) n Q, -(CH2) n When CHQR, -CHQR, or -CQ(R), (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R), or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl. In some embodiments, another subset of compounds of formula (I) is a compound in which R is C 5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR", -YR" and -R"M'R'; R2 and R3 are H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR” and -R * OR”, or R and R together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, wherein Q is selected from the group consisting of C 3-6 Carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R )S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) nand 5-14 membered heterocycloalkyl having one or more heteroatoms selected from N, O and S, selected from oxo (=O), OH, amino, mono- or di-alkylamino, and C(O)N(R)OR, -N(R)C(=NR)N(R), -N(R)C(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR)N(R), -N(OR)C(=CHR)N(R), -C(=NR)N(R), -C(=NR)R, -C(O)N(R)OR, and 5-14 membered heterocycloalkyl having one or more heteroatoms selected from N, O and S, selected from oxo (=O), OH, amino, mono- or di-alkylamino, and C(O)N(R)OR. 1-3 alkyl, where each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * selected from the group consisting of Y-R", -Y-R", and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * is independently C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m includes those selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0332] In some embodiments, another subset of compounds of formula (I) is: R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR”, or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, wherein Q is selected from the group consisting of C 3-6Carbocycle, 5-14 membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n and Q is a 5- to 14-membered heterocycle, and (i) R is -(CH) n Q, where n is 1 or 2, or (ii) R4 is -(CH2) n CHQR, where n is 1, or (iii) when R4 is -CHQR and -CQ(R)2, Q is either a 5-14 membered heteroaryl or an 8-14 membered heterocycloalkyl; Each R5 is independently 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, R * selected from the group consisting of YR″, YR″, and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * is independently C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m includes those selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0333] In some embodiments, another subset of compounds of formula (I) is: R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, YR”, and R* OR" or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, wherein Q is selected from the group consisting of C 3-6 Carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R), and each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, R * selected from the group consisting of YR″, YR″, and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * is independently C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m includes those selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0334] In some embodiments, another subset of compounds of formula (I) is: R1 is C5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 2-14 Alkyl, C 2-14 Alkenyl, -R * YR”, YR”, and R * OR”, or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is -(CH2) n Q or -(CH2) n CHQR, where Q is -N(R)2 and n is selected from 3, 4, and 5; Each R5 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, and aryl and heteroaryl groups; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * selected from the group consisting of Y-R", -Y-R", and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * is independently C 1-12 Alkyl and C 1-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m includes those selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0335] In some embodiments, another subset of compounds of formula (I) is: R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR", -YR", and -R"M'R'; R2 and R3 are independently C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, YR”, and R * OR”, or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is -(CH2) n Q, -(CH2) n selected from the group consisting of CHQR, -CHQR, and CQ(R)2, where Q is -N(R)2 and n is selected from 1, 2, 3, 4, and 5; Each R5 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, and aryl and heteroaryl groups; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * selected from the group consisting of Y-R", -Y-R", and H; Each R" is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * is independently C 1-12 Alkyl and C 1-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m includes those selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0336] In some embodiments, a subset of compounds of formula (I) are those of formula (IA): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M'; R is unsubstituted C 1-3 Alkyl, or -(CH2) nQ, where Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; and R and R are independently H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0337] In some embodiments, a subset of compounds of formula (I) are those of formula (II): [ka] or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; M is a bond or M'; R is an unsubstituted C 1-3 Alkyl, or -(CH2) n Q, where n is 2, 3, or 4, and Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; and R and R are independently H, C 1-14 Alkyl, and C 2-14 alkenyl. In some embodiments, a subset of compounds of Formula (I) are those of Formula (IIa), (IIb), (IIc), or (IIe): [ka] or a salt or isomer thereof, wherein R4 is as described herein.

[0338] In some embodiments, a subset of compounds of formula (I) are those of formula (IId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, and R2-R6 are as described herein. For example, each of R2 and R3 is C 5-14 Alkyl and C 5-14 alkenyl. In some embodiments, a subset of compounds of Formula (I) are those of Formula (IIa), (IIb), (IIc), or (IIe): [ka] or a salt or isomer thereof, wherein R4 is as defined herein.

[0339] In some embodiments, a subset of compounds of formula (I) are those of formula (IId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, and R2-R6 are as described herein. For example, each of R2 and R3 can independently be C 5-14 Alkyl and C 5-14 alkenyl. In some embodiments, the compound of Formula (I) is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] In a further embodiment, the compound of formula (I) is selected from the group consisting of: [ka] In some embodiments, the compound of Formula (I) is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and their salts and isomers. In some embodiments, the nanoparticles comprise the following compounds: [ka] or salts and isomers thereof.

[0340] In some embodiments, the present disclosure features a nanoparticle composition that includes a lipid component that includes a compound described herein (e.g., of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId), or (IIe)).

[0341] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional components in a vaccine composition may vary depending on the identity, size, and / or condition of the subject being treated, and further depending on 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.

[0342] In some embodiments, an RNA vaccine composition may include a polynucleotide described herein formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC, and PEG2000-DMG, the buffer trisodium citrate, sucrose, and water for injection. As a non-limiting example, a composition may include: 2.0 mg / mL of a drug (e.g., a polynucleotide encoding an H10N8 influenza virus), 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.

[0343] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm, or 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm. Flagellin is a monomeric protein of approximately 500 amino acids that polymerizes to form flagella in association with bacterial movement. Flagellin is expressed by various flagellated bacteria (e.g., Salmonella typhimurium) and non-flagellated bacteria (e.g., Escherichia coli). Flagellin detection by cells of the innate immune system (dendritic cells, macrophages, etc.) is mediated by Toll-like receptor 5 (TLR5) and Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and NLRs have been identified as playing a role in activating innate and adaptive immune responses. Therefore, flagellin provides an adjuvant effect in vaccines.

[0344] 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.

[0345] As used herein, a flagellin polypeptide refers to a full-length flagellin protein, an immunogenic fragment thereof, and a peptide having at least 50% sequence identity to the flagellin protein or an immunogenic fragment thereof. Exemplary flagellin proteins include flagellins from Salmonella typhi (UniPro Accession Number: Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonella choleraesuis (Q6V2X8). 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 an immunogenic fragment thereof.

[0346] In some embodiments, the flagellin polypeptide is an immunogenic fragment. The 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 substituted with other amino acids. For example, an antigen polypeptide may 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 region 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 of flagellin, or the entire hinge region. In other embodiments, the immunogenic fragment of flagellin is a 20-, 25-, 30-, 35-, or 40-amino acid C-terminal fragment of flagellin.

[0347] The flagellin monomer is formed by domains D0-D3. The stem, D0 and D1, consist of a long tandem α-helix and are highly conserved among different bacteria. The D1 domain contains several stretches of amino acids useful for TLR5 activation. The entire D1 domain or one or more active regions within the 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. Within the 13 amino acids in the 88-100 region, at least six substitutions are possible between Salmonella flagellins and other flagellins that still retain TLR5 activation. Thus, immunogenic fragments of flagellin include flagellin-like sequences that activate TLR5 and contain a 13-amino acid motif that is 53% or more identical to the Salmonella sequence in the 88-100 region of FliC (LQRVRELAVQSAN; SEQ ID NO: 428).

[0348] 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 may comprise, for example, one, two, three, four, five, six, or more flagellin polypeptides linked to one, two, three, four, five, six, or more antigenic polypeptides. When two or more flagellin polypeptides and / or two or more antigenic polypeptides are linked, such a construct may be referred to as a "multimer."

[0349] The components of the fusion protein may be directly linked to each other or may be linked via a linker. For example, the linker may be an amino acid linker. The amino acid linker encoded by the RNA (e.g., mRNA) vaccine that links the components of the fusion protein may include, for example, at least one member selected from the group consisting of lysine residues, glutamic acid residues, serine residues, and arginine residues. 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. In other embodiments, the RNA (e.g., mRNA) vaccine comprises at least two separate RNA polynucleotides, one encoding one or more antigen polypeptides and the other encoding a flagellin polypeptide. The at least two RNA polynucleotides may be co-formulated in a carrier, such as a lipid nanoparticle.

[0350] Liposomes, lipoplexes and lipid nanoparticles The RNA vaccines of the present invention may be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, the RNA vaccine comprises one or more RNA polynucleotides containing one or more open reading frames encoding one or more of the HCMV antigen polypeptides gB, gH, gL, UL128, UL130, and UL131. In some embodiments, all of the RNA polynucleotide components of the vaccine are formulated in the same liposome, lipoplex, or lipid nanoparticle. In other embodiments, one or more of the RNA polynucleotide components of the vaccine are formulated in different liposomes, lipoplexes, or lipid nanoparticles. In other embodiments, each of the RNA polynucleotide components of the vaccine is formulated in a different liposome, lipoplex, or lipid nanoparticle. In some embodiments, the RNA vaccine comprises RNA polynucleotides encoding gB, gH, gL, UL128, UL130, and UL131. The RNA polynucleotides encoding gB, gH, gL, UL128, UL130, and UL131 may be formulated in one or more liposomes, lipoplexes, or lipid nanoparticles. In certain embodiments, the RNA polynucleotides encoding gB, gH, gL, UL128, UL130, and UL131 are all contained in the same liposome, lipoplex, or lipid nanoparticle.

[0351] In some embodiments, the pharmaceutical composition of the RNA vaccine includes liposomes. Liposomes are artificially prepared vesicles, primarily composed of lipid bilayers, that can be used as delivery vehicles for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve liposome adhesion to unhealthy tissues or to activate events such as endocytosis. Liposomes can also contain low or high pH to improve delivery of pharmaceutical formulations.

[0352] The formation of liposomes may depend on physicochemical properties, including but not limited to, the encapsulated pharmaceutical agent and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any further processes involved during application and / or delivery of the vesicles, the optimized size, polydispersity and shelf life of the vesicles for the intended use, and the possibility of batch-to-batch reproducibility and large-scale production of a safe and efficient liposome product.

[0353] As a non-limiting example, liposomes such as synthetic membrane vesicles can be prepared by the methods, apparatus, and devices described in U.S. Patent Application Publication Nos. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373, and US20130183372, the contents of each of which are incorporated herein by reference in their entirety.

[0354] In some embodiments, the pharmaceutical compositions described herein may comprise liposomes capable of delivering small molecule drugs, such as, but not limited to, liposomes formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (U.S. Patent Application Publication No. 20100324120; incorporated herein by reference in its entirety), and DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[0355] In some embodiments, the pharmaceutical compositions described herein may comprise liposomes, such as, but not limited to, liposomes formed from the synthesis of stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have previously been described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (Wheeler et al., Gene Therapy. 1999 6:271-281; ​​Zhang et al., Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; U.S. Patent Application Publication No. US20130122104 (all of which are incorporated herein in their entirety). The original manufacturing method by Wheeler et al. was the detergent dialysis method, which was later improved by Jeffs et al. and is called the spontaneous vesicle formation method. Liposome formulations consist of three to four lipid components in addition to the polynucleotide. By way of example, liposomes may contain, but are not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al.As another example, a particular liposome formulation may include, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, which may include 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

[0356] In some embodiments, the liposome formulation may contain about 25.0% to about 40.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol. In preferred embodiments, the formulation may contain a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, and 43.5%. In some embodiments, the formulation may contain about 5.0% to about 10.0% DSPC and / or about 7.0% to about 15.0% DSPC.

[0357] In some embodiments, the pharmaceutical composition may comprise liposomes, which can be formed to deliver a polynucleotide that can encode at least one immunogen (antigen) or any other polypeptide of interest. The RNA vaccine may be encapsulated by liposomes and / or contained in an aqueous core that can be subsequently encapsulated by liposomes (see International Application Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and U.S. Patent Application Publication Nos. US20130189351, US20130195969 and US20130202684, the contents of each of which are incorporated herein by reference in their entirety).

[0358] In another embodiment, liposome can be formulated for targeted delivery.For example, liposome can be formulated for targeted delivery to liver.The liposome used for targeted delivery can include but is not limited to the liposome and the method for producing the liposome described in US Patent Application Publication No. US20130195967, the content of which is incorporated herein by reference in its entirety.

[0359] In another embodiment, a polynucleotide capable of encoding an immunogen (antigen) can be formulated in a cationic oil-in-water emulsion, where the emulsion particles comprise an oil core and cationic lipids capable of interacting with the polynucleotide to anchor the molecule to the emulsion particle (see International Application No. WO2012006380, which is incorporated herein by reference in its entirety).

[0360] In some embodiments, the RNA vaccine may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which a hydrophilic phase is dispersed. As a non-limiting example, the emulsion may be produced by the method described in International Application No. WO201087791, the contents of which are incorporated herein by reference in their entirety.

[0361] In another embodiment, the lipid formulation may include at least a cationic lipid, a lipid capable of enhancing transfection, and at least one lipid comprising a hydrophilic head group attached to the lipid moiety (International Application No. WO2011076807 and U.S. Patent Application Publication No. 20110200582; the contents of each of which are incorporated herein by reference in their entirety). In another embodiment, the polynucleotide encoding the immunogen may be formulated into lipid vesicles that may have crosslinks between functionalized lipid bilayers (see U.S. Patent Application Publication No. 20120177724, the contents of which are incorporated herein by reference in their entirety).

[0362] In some embodiments, polynucleotides may be formulated in liposomes, as described in International Application No. WO2013086526, the contents of which are incorporated herein by reference in their entirety. RNA vaccines may be encapsulated in liposomes using a reverse pH gradient and / or optimized internal buffer composition, as described in International Application No. WO2013086526, the contents of which are incorporated herein by reference in their entirety.

[0363] In some embodiments, the RNA vaccine pharmaceutical composition may be formulated in liposomes, including, 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).

[0364] In some embodiments, the cationic lipid may be a low molecular weight cationic lipid such as those described in U.S. Patent Application No. 20130090372, the contents of which are incorporated herein by reference in their entirety.

[0365] In some embodiments, the RNA vaccines may be formulated into lipid vesicles that may have crosslinks between functionalized lipid bilayers.

[0366] In some embodiments, the RNA vaccine may be formulated in a liposome containing a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to phosphate in the RNA (N:P ratio) of 1:1 to 20:1, as described in International Application No. WO2013006825, the entirety of which is incorporated herein. In other embodiments, the liposome may have an N:P ratio greater than 20:1 or less than 1:1.

[0367] In some embodiments, the RNA vaccine may be formulated in a lipid-polycation complex. Formation of the lipid-polycation complex may be achieved by methods known in the art and / or as described in U.S. Patent Application Publication No. 20120178702, the entire contents of which are incorporated herein by reference. Non-limiting examples of polycations include cationic peptides or polypeptides such as polylysine, polyornithine, and / or polyarginine, as well as the cationic peptides described in International Application No. WO2012013326 or U.S. Patent Application Publication No. US20130142818, each of which is incorporated herein by reference in its entirety. In another embodiment, the RNA vaccine may be formulated in a lipid-polycation complex, which may further include a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0368] In some embodiments, the RNA vaccine may be formulated in an amino alcohol lipidoid. The amino alcohol lipidoid that can be used in the present invention may be prepared by the method described in U.S. Patent No. 8,450,298, the entire contents of which are incorporated herein by reference.

[0369] Liposome formulation can be influenced by biophysical parameters, including but not limited to, the selection of cationic lipid components, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and size.In one example by Semple et al. (Semple et al., Nature Biotech.2010 28:172-176; its entirety is incorporated herein by reference), liposome formulation is composed of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol and 1.4% PEG-DMA.In another example, by changing the composition of cationic lipid, siRNA can be more effectively delivered to various antigen-presenting cells (Basha et al., Mol Ther.2011 19:2186-2200; its entirety is incorporated herein by reference). In some embodiments, the liposome formulation may comprise about 35 to about 45% cationic lipid, about 40 to about 50% cationic lipid, about 50 to about 60% cationic lipid, and / or about 55 to about 65% cationic lipid. In some embodiments, the lipid to mRNA ratio in the liposome may be about 5:1 to about 20:1, about 10:1 to about 25:1, about 15:1 to about 30:1, and / or at least 30:1.

[0370] In some embodiments, the ratio of PEG in a lipid nanoparticle (LNP) formulation may be increased or decreased, and / or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the LNP formulation. As a non-limiting example, an LNP formulation may contain a lipid molar ratio of about 0.5% to about 3.0%, about 1.0% to about 3.5%, about 1.5% to about 4.0%, about 2.0% to about 4.5%, about 2.5% to about 5.0%, and / or about 3.0% to 6.0% PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In another embodiment, 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.

[0371] In some embodiments, the RNA vaccine may be formulated in lipid nanoparticles as described in International Application No. WO2012170930, the contents of which are incorporated herein by reference in their entirety.

[0372] In some embodiments, the RNA vaccine formulation containing a polynucleotide is a nanoparticle that may contain at least one lipid.The lipid 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, PEGylated lipids, and aminoalcohol lipids.In another aspect, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids.The aminoalcohol cationic lipid may be the lipid described in U.S. Patent Application Publication No. US20130150625, the entire contents of which are incorporated herein by reference, and / or the lipid produced by the method 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 in U.S. Patent Application Publication No. 20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 ... -[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 of U.S. Patent Application Publication No. 20130150625); 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 U.S. Patent Application Publication No. 20130150625); or a pharmaceutically acceptable salt or stereoisomer thereof.

[0373] Lipid nanoparticle formulations typically contain lipids, particularly ionizable cationic lipids such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DL1-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), as well as neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids.

[0374] 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 about 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol; 0.5-15% PEG-lipid).

[0375] In some embodiments, the formulation comprises about 25% to about 75% on a molar basis, e.g., about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50%, or about 40% on a molar basis, of a cationic lipid selected from 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).

[0376] In some embodiments, the formulation contains about 0.5% to about 15% neutral lipid on a molar basis, e.g., about 3% to about 12%, about 5% to about 10%, or about 15%, about 10%, or about 7.5%. Exemplary neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation contains about 5% to about 50% sterol on a molar basis (e.g., about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31%). An exemplary sterol is cholesterol. In some embodiments, the formulation comprises about 0.5% to about 20% PEG or PEG-modified lipid on a molar basis (e.g., about 0.5% to about 10%, a...

Claims

1. A messenger ribonucleic acid (mRNA) vaccine, comprising: (a) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an open reading frame (ORF) encoding the hCMV gH protein, a 3' UTR, and a poly A tail; (b) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV gL protein, a 3' UTR, and a poly A tail; (c) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV UL128 protein, a 3' UTR, and a poly A tail; (d) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV UL130 protein, a 3' UTR, and a poly A tail; (e) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV UL131 protein, a 3' UTR, and a poly A tail; and lipid nanoparticles Including, An mRNA vaccine, wherein 100% of the uracil nucleosides in the ORF of the mRNA polynucleotide of (b) to (e) are N1-methylpseudouridine.

2. The mRNA vaccine described in claim 1, wherein the lipid nanoparticles comprise an ionizable cationic lipid, a PEG-modified lipid, a sterol, and a neutral lipid.

3. The mRNA vaccine described in claim 2, wherein the lipid nanoparticles comprise 20 to 60 mol% ionizable cationic lipid, 0.5 to 15 mol% PEG-modified lipid, 25 to 55 mol% sterol, and 5 to 25% neutral lipid.

4. The mRNA vaccine described in claim 3, wherein the PEG-modified lipid is PEG-DMG2000, the sterol is cholesterol, and the neutral lipid is disteroylphosphatidylcholine (DSPC).

5. The lipid nanoparticles are represented by the formula (I): 【Chemistry 1】 or a salt thereof, wherein R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, and —R″M′R′; R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, and C 2-14 alkenyl; R 4 is —(CH 2 ) n Q, where Q is —OR and n is selected from 1, 2, 3, 4, and 5; R 5 is H; R 6 is H; M and M′ are independently selected from —C(O)O— and —OC(O)—; R 7 is H; R is H; R' is selected from the group consisting of C 1-18 alkyl, and C 2-18 alkenyl; R" is selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; The mRNA vaccine of claim 4.

6. The lipid nanoparticles comprising Compound 25: 【Chemistry 2】 The mRNA vaccine of claim 5, comprising:

7. The mRNA vaccine described in claim 1, wherein the hCMV gH protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

59.

8. The mRNA vaccine described in claim 1, wherein the hCMV gL protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

61.

9. The mRNA vaccine described in claim 1, wherein the hCMV UL128 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

63.

10. The mRNA vaccine described in claim 1, wherein the hCMV UL130 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

65.

11. The mRNA vaccine described in claim 1, wherein the hCMV UL131 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

67. (f) an mRNA polynucleotide having, from 5' to 3', a 5'UTR, an ORF encoding hCMV gB protein, a 3'UTR, and a poly A tail. further comprising (f) 100% of the uracil nucleosides in the ORF of the mRNA polynucleotide are N1-methylpseudouridine; The mRNA vaccine of claim 1.

13. (a) the hCMV gH protein comprises the amino acid sequence of SEQ ID NO:59; (b) the hCMV gL protein comprises the amino acid sequence of SEQ ID NO: 61; (c) the hCMV UL128 protein comprises the amino acid sequence of SEQ ID NO: 63; (d) the hCMV UL130 protein comprises the amino acid sequence of SEQ ID NO: 65; and (e) the hCMV UL131 protein comprises the amino acid sequence of SEQ ID NO: 67; The mRNA vaccine of claim 1. (f) an mRNA polynucleotide having, from 5' to 3', a 5'UTR, an ORF encoding hCMV gB protein, a 3'UTR, and a poly A tail. further comprising the hCMV gB protein comprises the amino acid sequence of SEQ ID NO:69; (f) 100% of the uracil nucleosides in the ORF of the mRNA polynucleotide are N1-methylpseudouridine; The mRNA vaccine of claim 13.

15. (a) the hCMV gH protein comprises the amino acid sequence of SEQ ID NO:59; (b) the hCMV gL protein comprises the amino acid sequence of SEQ ID NO: 61; (c) the hCMV UL128 protein comprises the amino acid sequence of SEQ ID NO: 63; (d) the hCMV UL130 protein comprises the amino acid sequence of SEQ ID NO: 65; and (e) the hCMV UL131 protein comprises the amino acid sequence of SEQ ID NO: 67; The mRNA vaccine of claim 6.

16. The lipid nanoparticles comprising Compound 25: 【Transformation 3】 The mRNA vaccine of claim 14, comprising:

17. An mRNA vaccine for use in inducing an immune response to hCMV in a subject, comprising: (a) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an open reading frame (ORF) encoding the hCMV gH protein, a 3' UTR, and a poly A tail; (b) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV gL protein, a 3' UTR, and a poly A tail; (c) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV UL128 protein, a 3' UTR, and a poly A tail; (d) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV UL130 protein, a 3' UTR, and a poly A tail; (e) an mRNA polynucleotide having, from 5' to 3', a 5' UTR, an ORF encoding the hCMV UL131 protein, a 3' UTR, and a poly A tail; and lipid nanoparticles Including, An mRNA vaccine, wherein 100% of the uracil nucleosides in the ORF of the mRNA polynucleotide of (a) to (e) are N1-methylpseudouridine.

18. (a) the hCMV gH protein comprises the amino acid sequence of SEQ ID NO:59; (b) the hCMV gL protein comprises the amino acid sequence of SEQ ID NO: 61; (c) the hCMV UL128 protein comprises the amino acid sequence of SEQ ID NO: 63; (d) the hCMV UL130 protein comprises the amino acid sequence of SEQ ID NO: 65; and (e) the hCMV UL131 protein comprises the amino acid sequence of SEQ ID NO: 67; 18. An mRNA vaccine for use according to claim 17. (f) an mRNA polynucleotide having, from 5' to 3', a 5'UTR, an ORF encoding hCMV gB protein, a 3'UTR, and a poly A tail. further comprising The hCMV gB protein comprises the amino acid sequence of SEQ ID NO:

69. (f) 100% of the uracil nucleosides in the ORF of the mRNA polynucleotide are N1-methylpseudouridine; 18. An mRNA vaccine for use according to claim 17.

20. The lipid nanoparticle comprising compound 25: 【Chemistry 4】 19. The mRNA vaccine for use according to claim 18, comprising:

21. The lipid nanoparticle comprising compound 25: 【Transformation 5】 20. The mRNA vaccine for use according to claim 19, comprising:

22. An mRNA vaccine for use as described in claim 20, wherein the subject produces antibodies that neutralize hCMV.

23. An mRNA vaccine for use as described in claim 21, wherein the subject produces antibodies that neutralize hCMV.

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