Polynucleotides encoding norovirus VP1 antigens and uses thereof

Abstract

The present invention relates to nucleic acids and related immunogenic polypeptides for the prevention or treatment of infectious diseases. In particular, the nucleic acids and immunogenic polypeptides provide utility for the prophylactic prevention of norovirus infection in mammals. The present invention further relates to nucleic acid-based vaccine compositions and kits of parts comprising the vaccine compositions, as well as methods of treatment and medical uses involving the nucleic acids, vaccine compositions, and kits of parts.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 500,749, filed May 8, 2023, the entire contents of which are incorporated herein by reference.

[0002] Reference to an electronically submitted sequence listing The contents of the Electronic Sequence Listing (25691-WO-PCT_SL.xml; size: 6 kilobytes; created on August 24, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates generally to messenger ribonucleic acid (mRNA) compositions and vaccines and methods of using said compositions and vaccines for the treatment of infectious diseases such as norovirus. [Background technology]

[0004] Norovirus, also known as "Norwalk virus," was named after an outbreak of acute gastroenteritis at an elementary school in Norwalk, Ohio. Symptoms of norovirus infection appear within 12 to 48 hours of exposure and are characterized by nausea, vomiting, persistent diarrhea, lethargy, myasthenia, muscle pain, headache, cough, and / or fever. While severe illness is rare and norovirus infections are generally self-limited, norovirus remains one of the most common causes of acute gastroenteritis in people of all ages. Approximately 700 million norovirus infections are reported worldwide each year, with children under the age of five accounting for approximately 28.5% of all annual infections.

[0005] Taxonomically, noroviruses are a diverse group of single-stranded, positive-sense RNA, non-enveloped viruses belonging to the Caliciviridae family. Noroviruses contain a linear, non-segmented, positive-sense RNA genome of approximately 7.5 kilobases that encodes a large polyprotein, a major structural protein (VP1) of approximately 58–60 kDa, and a minor capsid protein (VP2), which are cleaved into six smaller nonstructural proteins (NS1 / 2–NS7) by a viral 3C-like protease (NS6). Noroviruses can be genetically classified into at least seven distinct genotype groups (GI, GII, GIII, GIV, GV, GVI, and GVII), which can be further divided into distinct genetic clusters or genotypes. Most noroviruses that infect humans belong to genotype groups GI and GII. Noroviruses from genotype group II, genotype 4 (abbreviated as GII.4) account for the majority of gastroenteritis outbreaks in adults, often causing pandemics worldwide.

[0006] Norovirus is highly contagious and transmissible; as few as five virions may be sufficient to cause an infection. Norovirus is typically spread via contaminated water or food by the fecal-oral route. However, airborne transmission via aerosolized virus has been documented. Therefore, preventive measures such as frequent hand washing, disinfecting surfaces, and avoiding contact with infected individuals may help reduce the risk of infection.

[0007] Currently, there are no specific preventive or therapeutic treatments for norovirus infections. Treatment is limited to symptomatic treatment of acute infections. Therefore, there is a strong need for additional therapeutic measures for norovirus infections. An object of the present invention is to provide a pharmaceutical composition for the preventive and therapeutic treatment of norovirus infections. In particular, several aspects of the present invention provide a norovirus vaccine that can be administered to mammals for the prevention and / or treatment of norovirus infections. Summary of the Invention

[0008] The present disclosure provides mRNA polynucleotides, pharmaceutical vaccine compositions, and kits containing them. The mRNA polynucleotides, vaccine compositions, and kits are useful for the preventive and therapeutic treatment of norovirus infections. The present disclosure also provides vectors and host cells containing the mRNA polynucleotides, methods for producing the pharmaceutical vaccine compositions, and methods for using the pharmaceutical vaccine compositions for the treatment of norovirus infections.

[0009] In one aspect, the disclosure provides an mRNA polynucleotide encoding a Norovirus VP1 polypeptide, wherein the mRNA polynucleotide is derived from a Norovirus selected from the group consisting of GII.4, GI.1, GII.2, GII.3, and GII.6 Norovirus, and wherein at least one uridine residue in the mRNA polynucleotide is replaced with N1-methylpseudouridine.

[0010] In one aspect, the present disclosure provides an mRNA polynucleotide encoding a Norovirus VP1 polypeptide, wherein the mRNA polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein at least one uridine residue in the mRNA polynucleotide is replaced with N1-methylpseudouridine. In certain embodiments, the Norovirus VP1 polypeptide is an antigenic polypeptide.

[0011] In one aspect, the present disclosure provides an mRNA polynucleotide encoding a Norovirus VP1 polypeptide, wherein the mRNA polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5, and wherein at least one uridine residue in the mRNA polynucleotide is replaced with N1-methylpseudouridine.

[0012] In some embodiments, the mRNA polynucleotide is derived from a Norovirus selected from the group consisting of GII.4, GI.1, GII.2, GII.3, and GII.6 Norovirus.

[0013] In some embodiments, the mRNA polynucleotide further comprises a heterologous 5' untranslated region (UTR) and a heterologous 3' UTR.

[0014] In some embodiments, the 5'UTR comprises the polynucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5'UTR further comprises the 5' cap structure N7-methyl-G(3'Ome)ppp.

[0015] In some embodiments, the 3'UTR comprises a poly(A) sequence and / or a poly(C) sequence, in which the poly(A) sequence comprises 10 to 200 adenosine nucleotides (SEQ ID NO: 19) and / or the poly(C) sequence comprises 10 to 200 cytosine nucleotides (SEQ ID NO: 20).

[0016] In some embodiments, the poly(A) sequence comprises 10 to 100 adenosine nucleotides (SEQ ID NO: 21). In some embodiments, the poly(A) sequence comprises 10 to 80 adenosine nucleotides (SEQ ID NO: 22). In some embodiments, the poly(A) sequence comprises 50 to 70 adenosine nucleotides (SEQ ID NO: 23). In certain embodiments, the poly(A) sequence comprises 80 adenine nucleotides (SEQ ID NO: 24).

[0017] In some embodiments, the poly(C) sequence comprises 10 to 100 cytosine nucleotides (SEQ ID NO: 25). In some embodiments, the poly(C) sequence comprises 20 to 70 cytosine nucleotides (SEQ ID NO: 26). In some embodiments, the poly(C) sequence comprises 20 to 60 cytosine nucleotides (SEQ ID NO: 27). In some embodiments, the poly(C) sequence comprises 10 to 40 cytosine nucleotides (SEQ ID NO: 28).

[0018] In some embodiments, the 3' UTR comprises the polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In certain embodiments, the 3' UTR comprises the polynucleotide sequence of SEQ ID NO: 7. In certain embodiments, the 3' UTR comprises the polynucleotide sequence of SEQ ID NO: 8.

[0019] In certain embodiments, the mRNA polynucleotide comprises, from 5' to 3', a 5' UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a 3' UTR comprising the polynucleotide sequence of SEQ ID NO: 7, wherein the 5' UTR further comprises a 5' cap structure N7-methyl-G(3'OMe)ppp, and all uridine residues in the polynucleotide encoding the Norovirus VP1 polypeptide are replaced with N1-methylpseudouridine. In certain embodiments, the Norovirus VP1 polypeptide is an antigenic polypeptide.

[0020] In certain embodiments, the mRNA polynucleotide comprises, from 5' to 3', a 5'UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5; and a 3'UTR comprising the polynucleotide sequence of SEQ ID NO: 7, wherein the 5'UTR further comprises a 5' cap structure N7-methyl-G(3'Ome)ppp, and all uridine residues in the polynucleotide encoding the Norovirus VP1 polypeptide are replaced with N1-methylpseudouridine.

[0021] In certain embodiments, the mRNA polynucleotide comprises, from 5' to 3', a 5' UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a 3' UTR comprising the polynucleotide sequence of SEQ ID NO: 8, wherein the 5' UTR further comprises a 5' cap structure N7-methyl-G(3'OMe)ppp, and all uridine residues in the polynucleotide encoding the Norovirus VP1 polypeptide are replaced with N1-methylpseudouridine. In certain embodiments, the Norovirus VP1 polypeptide is an antigenic polypeptide.

[0022] In certain embodiments, the mRNA polynucleotide comprises, from 5' to 3', a 5'UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5; and a 3'UTR comprising the polynucleotide sequence of SEQ ID NO: 8, wherein the 5'UTR further comprises a 5' cap structure N7-methyl-G(3'OMe)ppp, and all uridine residues in the polynucleotide encoding the Norovirus VP1 polypeptide are replaced with N1-methylpseudouridine.

[0023] In some embodiments, the mRNA polynucleotide comprises at least one histone stem-loop structure.

[0024] In some embodiments, at least 50% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 75% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 85% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 90% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 95% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 96% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 97% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 98% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In some embodiments, at least 99% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine. In certain embodiments, 100% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine.

[0025] In another aspect, the present disclosure provides a combination of one or more mRNA polynucleotides described herein. In certain embodiments, the one or more mRNA polynucleotides are 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 mRNA polynucleotides described herein. In certain embodiments, the one or more mRNA polynucleotides are at least 1, at least 2, at least 3, at least 4, at least 5, or 5 mRNA polynucleotides described herein.

[0026] In another aspect, the present disclosure provides a composition comprising at least one mRNA polynucleotide encoding a Norovirus VP1 polypeptide described herein and a pharmaceutically acceptable carrier.

[0027] In another aspect, the present disclosure provides a composition comprising at least two mRNA polynucleotides encoding a Norovirus VP1 polypeptide described herein and a pharmaceutically acceptable carrier.

[0028] In another aspect, the present disclosure provides a composition comprising at least three mRNA polynucleotides encoding a Norovirus VP1 polypeptide described herein and a pharmaceutically acceptable carrier.

[0029] In another aspect, the present disclosure provides a composition comprising at least four mRNA polynucleotides encoding Norovirus VP1 polypeptides described herein and a pharmaceutically acceptable carrier.

[0030] In another aspect, the present disclosure provides a composition comprising at least five mRNA polynucleotides encoding a Norovirus VP1 polypeptide described herein and a pharmaceutically acceptable carrier. In some embodiments, the at least five mRNA polynucleotides are derived from GII.4, GI.1, GII.2, GII.3, and GII.6 Noroviruses. In some embodiments, the composition comprises five mRNA polynucleotides, each derived from one of GII.4, GI.1, GII.2, GII.3, and GII.6 Noroviruses, and a pharmaceutically acceptable carrier.

[0031] In another aspect, the disclosure provides a composition comprising five mRNA polynucleotides encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier, wherein each of the five mRNA polynucleotides comprises, from 5' to 3', a 5' UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a 3' UTR comprising the polynucleotide sequence of SEQ ID NO: 7; The 5'UTR further comprises a 5' cap structure N7-methyl-G(3'OMe)ppp, and the composition comprises each of SEQ ID NOS: 1-5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein all uridine residues in the five mRNA polynucleotides in each of SEQ ID NOS: 1-5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, are replaced with N1-methylpseudouridines. In certain embodiments, a Norovirus VP1 polypeptide is an antigenic polypeptide.

[0032] In another aspect, the present disclosure provides a composition comprising five mRNA polynucleotides encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier, wherein each of the five mRNA polynucleotides comprises, from 5' to 3', a 5' UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5; and a 3' UTR comprising the polynucleotide sequence of SEQ ID NO: 7, wherein the 5' UTR further comprises the 5' cap structure N7-methyl-G(3'OMe)ppp; and wherein the composition comprises each of SEQ ID NOs: 1-5, wherein all uridine residues in the five mRNA polynucleotides are replaced with N1-methylpseudouridine in each of SEQ ID NOs: 1-5.

[0033] In another aspect, the disclosure provides a composition comprising five mRNA polynucleotides encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier, wherein each of the five mRNA polynucleotides comprises, from 5' to 3', a 5' UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a 3' UTR comprising the polynucleotide sequence of SEQ ID NO: 8; The 5'UTR further comprises a 5' cap structure N7-methyl-G(3'OMe)ppp, and the composition comprises each of SEQ ID NOS: 1-5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein all uridine residues in the five mRNA polynucleotides in each of SEQ ID NOS: 1-5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, are replaced with N1-methylpseudouridines. In certain embodiments, a Norovirus VP1 polypeptide is an antigenic polypeptide.

[0034] In another aspect, the present disclosure provides a composition comprising five mRNA polynucleotides encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier, wherein each of the five mRNA polynucleotides comprises, from 5' to 3', a 5' UTR comprising the polynucleotide sequence of SEQ ID NO: 6; a polynucleotide encoding a Norovirus VP1 polypeptide, the polynucleotide being selected from the group consisting of SEQ ID NOs: 1-5; and a 3' UTR comprising the polynucleotide sequence of SEQ ID NO: 8, wherein the 5' UTR further comprises the 5' cap structure N7-methyl-G(3'OMe)ppp; and wherein the composition comprises each of SEQ ID NOs: 1-5, wherein all uridine residues in the five mRNA polynucleotides are replaced with N1-methylpseudouridine in each of SEQ ID NOs: 1-5.

[0035] In some embodiments, the composition further comprises one or more additional mRNA polynucleotides derived from a norovirus selected from the group consisting of genogroup I norovirus, genogroup II norovirus, genogroup III norovirus, genogroup IV norovirus, and genogroup V norovirus. In some embodiments, the norovirus VP1 polypeptide of the composition is derived from a genogroup I norovirus or a genogroup II norovirus. In some embodiments, the norovirus VP1 polypeptide of the composition is derived from a genotype group selected from the group consisting of GII.4, GI.1, GII.2, GII.3, and GII.6 norovirus. In some embodiments, the norovirus VP1 polypeptide of the composition is selected from the group consisting of SEQ ID NOs: 14-18 and sequences having at least 70% identity thereto.

[0036] In another aspect, the present disclosure provides a composition comprising at least one mRNA polynucleotide encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises at least one, at least two, at least three, at least four, or at least five mRNA polynucleotides encoding a Norovirus VP1 polypeptide.

[0037] In some embodiments, each mRNA polynucleotide is separately encapsulated in a lipid nanoparticle (LNP) that comprises one or more cationic or polycationic compounds.

[0038] In some embodiments, each mRNA polynucleotide is co-encapsulated in an LNP that includes one or more cationic or polycationic compounds.

[0039] In some embodiments, each mRNA polynucleotide encapsulated in the LNP is present in equal amounts.

[0040] In some embodiments, each mRNA polynucleotide encapsulated in the LNP is not present in equal amounts.

[0041] In some embodiments, the LNPs comprise one or more of a cationic lipid, a sterol, a phospholipid, and a polyethylene glycol-lipid, while in other embodiments, the LNPs comprise a cationic lipid, a sterol, a phospholipid, and a polyethylene glycol-lipid.

[0042] In some embodiments, the LNP comprises 34-59 mol% cationic lipid, 30-48 mol% sterol, 10-24 mol% phospholipid, and 1-2 mol% polyethylene glycol-lipid.

[0043] In some embodiments, the cationic lipid is [Table 1]

[0044] In some embodiments, the sterol is [ka] is.

[0045] In some embodiments, the phospholipid is [Table 2]

[0046] In some embodiments, the polyethylene glycol-lipid is [Table 3]

[0047] In some embodiments, the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

[0048] In some embodiments, the sterol is cholesterol.

[0049] In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0050] In some embodiments, the polyethylene glycol-lipid is dimyristoylglycerol-polyethylene glycol (DMG-PEG).

[0051] In some embodiments, the LNPs comprise 49-59 mol% (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine. In other embodiments, the LNPs comprise about 58 mol% (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine. In certain embodiments, the LNPs comprise about 49 mol% (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

[0052] In another aspect, the present disclosure provides a vaccine comprising an mRNA polynucleotide, combination, or composition described herein.

[0053] In some embodiments, the vaccine further comprises a pharmaceutically acceptable carrier. In some embodiments, the vaccine further comprises an adjuvant.

[0054] In some embodiments, the vaccine is monovalent, bivalent, trivalent, tetravalent, or pentavalent.

[0055] In some embodiments, the mRNA polynucleotides, combinations, compositions and / or vaccines described herein are immunogenic in a mammalian species.

[0056] In another aspect, the present disclosure provides a kit or kit-of-parts comprising an mRNA polynucleotide, combination, composition or vaccine as described herein, said kit or kit-of-parts may further comprise a liquid vehicle for solubilization and / or technical instructions providing information on the administration and dosage of the components.

[0057] In another aspect, the present disclosure provides an mRNA polynucleotide, combination, composition, vaccine or kit or kit-of-parts as described herein for use as a medicament.

[0058] In another aspect, the present disclosure provides an mRNA polynucleotide, combination, composition, vaccine, or kit or kit-of-parts as described herein for use in the manufacture of a medicament for the treatment or prevention of infection with Norovirus or a disorder associated with infection with Norovirus.

[0059] In another aspect, the present disclosure provides an mRNA polynucleotide, combination, composition, vaccine, or kit or kit-of-parts described herein for use in the treatment or prevention of infection with Norovirus or a disorder associated with infection with Norovirus.

[0060] In another aspect, the present disclosure provides an mRNA polynucleotide, combination, composition, vaccine or kit or kit-of-parts as described herein, wherein an effective amount of the active ingredients of the polynucleotide, combination, composition, vaccine or kit or kit-of-parts is administered by injection.

[0061] In another aspect, the present disclosure provides a method of treating or preventing a disorder, the method comprising administering to a subject in need thereof an effective amount of an mRNA polynucleotide, combination, composition, vaccine, or active ingredient of a kit or kit-of-parts described herein. In some embodiments, the disorder is a Norovirus infection or a disorder associated with a Norovirus infection.

[0062] In another aspect, the present disclosure provides vectors comprising one or more nucleic acids encoding the mRNA polynucleotides described herein.

[0063] In another aspect, the present disclosure provides a host cell comprising an mRNA polynucleotide, combination, or vector described herein.

[0064] In another aspect, the present disclosure provides polypeptides encoded by the mRNA polynucleotides described herein.

[0065] In another aspect, the disclosure provides a method for producing a virus-like particle (VLP) comprising one or more Norovirus VP1 polypeptides, the method comprising transcribing an mRNA polynucleotide, combination, or vector described herein in a recombinant nucleic acid expression system under conditions suitable for assembly of the expressed one or more Norovirus VP1 polypeptides into a VLP.

[0066] In another aspect, the present disclosure provides a VLP comprising one or more Norovirus VP1 polypeptides produced according to the methods described herein.

[0067] The above summary of the present technology is non-limiting; other features and advantages of the present technology will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0068] [Figure 1] FIG. 1 shows a 2% agarose E-gel of purified mRNA for five norovirus VP1-encoding sequences. [Figure 2A] Figures 2A-2E show plots representing interpolated endpoint titers detected in the sera of mice vaccinated with the test vaccines. Figure 2A shows the titers for GI.1. [Figure 2B] Figures 2A-2E show plots representing interpolated endpoint titers detected in the sera of mice vaccinated with the test vaccines. Figure 2B shows titers for GII.2. [Figure 2C] Figures 2A-2E show plots representing interpolated endpoint titers detected in the sera of mice vaccinated with the test vaccines. Figure 2C shows titers for GII.3. [Figure 2D] Figures 2A-2E show plots representing interpolated endpoint titers detected in the sera of mice vaccinated with the test vaccines. Figure 2D shows titers for GII.4. [Figure 2E] Figures 2A-2E show plots representing interpolated endpoint titers detected in the sera of mice vaccinated with the test vaccines, with Figure 2E showing titers for GII.6. [Figure 3A] Figures 3A-3E show plots depicting histo-blood group antigen (HBGA) blocking detected in the serum of mice vaccinated with the test vaccines. Figure 3A shows titers for GI.1. [Figure 3B] Figures 3A-3E show plots depicting histo-blood group antigen (HBGA) blocking detected in the sera of mice vaccinated with the test vaccines. Figure 3B shows titers for GII.2. [Figure 3C] Figures 3A-3E show plots depicting histo-blood group antigen (HBGA) blocking detected in the serum of mice vaccinated with the test vaccines, and Figure 3C shows titers for GII.3. [Figure 3D] FIG. 3D shows the titers for GII.4. [Figure 3E] Figures 3A-3E show plots depicting histo-blood group antigen (HBGA) blocking detected in the sera of mice vaccinated with the test vaccines, with Figure 3E showing titers for GII.6. [Figure 4A] Figures 4A-4E show plots representing interpolated endpoint titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 4A shows the titers for GI.1. [Figure 4B] Figures 4A-4E show plots representing interpolated endpoint titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 4B shows titers for GII.2. [Figure 4C] Figures 4A-4E show plots representing interpolated endpoint titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 4C shows titers for GII.3. [Figure 4D] Figures 4A-4E show plots representing interpolated endpoint titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 4D shows titers for GII.4. [Figure 4E] Figures 4A-4E show plots representing interpolated endpoint titers detected in the sera of NHPs vaccinated with the test vaccines, with Figure 4E showing titers for GII.6. [Figure 5A] Figures 5A-5E show plots depicting HBGA blocking titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 5A shows the titers for GI.1. [Figure 5B] Figures 5A-5E show plots depicting HBGA blocking titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 5B shows titers for GII.2. [Figure 5C] Figures 5A-5E show plots depicting HBGA blocking titers detected in the sera of NHPs vaccinated with the test vaccines. Figure 5C shows titers for GII.3. [Figure 5D]Figures 5A-5E show plots depicting HBGA blocking titers detected in the sera of NHPs vaccinated with the test vaccines, and Figure 5D shows titers for GII.4. [Figure 5E] Figures 5A-5E show plots depicting HBGA blocking titers detected in the sera of NHPs vaccinated with the test vaccines, with Figure 5E showing titers for GII.6. DETAILED DESCRIPTION OF THE INVENTION

[0069] The present disclosure provides mRNA polynucleotides, pharmaceutical vaccine compositions, and kits containing them. The mRNA polynucleotides, vaccine compositions, and kits are useful for the preventive and therapeutic treatment of norovirus infections. The present disclosure also provides vectors and host cells containing the mRNA polynucleotides, methods for producing the pharmaceutical vaccine compositions, and methods for using the pharmaceutical vaccine compositions for the treatment of norovirus infections.

[0070] definition Listed here are definitions of various terms used in this specification. These definitions apply to these terms as they appear throughout the specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature and laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry used herein are those well known and commonly used in the art.

[0072] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Furthermore, use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0073] As used herein, the term "about" in quantitative terms refers to plus or minus 10% of the value it modifies (if the value is not sub-dividable, such as the number of molecules or nucleotides, it is rounded up to the nearest integer).

[0074] All ranges disclosed herein are inclusive of the recited endpoints and are independently combinable (e.g., the range "50 mg to 500 mg" includes the endpoints 50 mg and 500 mg, and all intermediate values). The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values; they are sufficiently imprecise to include values ​​that approximate those ranges and / or values.

[0075] As used herein, the term "comprising" can include the embodiments of "consisting of" and "consisting essentially of." As used herein, the terms "comprise(s)," "include(s)," "having," "has," "may," "contain(s)," and variations thereof, are intended to be open-ended transitional phrases, terms, or words that require the presence of the specified components / steps and allow for the presence of others. However, such descriptions should also be interpreted as describing compositions or processes as "consisting of" and "consisting essentially of" the listed components, along with any acceptable carriers or fluids, allowing for the presence of only the specified components or compounds and excluding others.

[0076] Nucleic Acids / Polynucleotides The norovirus vaccines provided herein contain at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one norovirus antigenic polypeptide. In its broadest sense, the term "nucleic acid" includes any compound and / or substance containing a polymer of nucleotides. These polymers are referred to as "polynucleotides." Nucleic acids (also referred to as polynucleotides) can 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 (including LNA with a β-D-ribonucleotide configuration, α-LNA (a diastereomer of LNA) with an α-L-ribonucleotide configuration, 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.

[0077] In some embodiments, the polynucleotides of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (a naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will understand that, unless otherwise specified, the polynucleotide sequences described in this application describe "T" in a representative DNA sequence, but when the sequence represents RNA, "T" is replaced with "U."

[0078] The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly(A) tail. The polynucleotides of the present disclosure can function as mRNAs but can be distinguished from wild-type mRNAs in their functional and / or structural design features that help overcome the existing problem of effective polypeptide expression using nucleic acid-based therapeutics.

[0079] In some embodiments, the RNA polynucleotides of the Norovirus vaccine encode 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 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 polynucleotides of the Norovirus vaccine encode antigenic polypeptides derived from a single Norovirus genotype, i.e., the vaccine is "monovalent." In some embodiments, the RNA polynucleotides of the norovirus vaccine encode antigenic polypeptides from two different norovirus genotypes, i.e., the vaccine is "bivalent." In some embodiments, the RNA polynucleotides of the norovirus vaccine encode antigenic polypeptides from three different norovirus genotypes, i.e., the vaccine is "trivalent." In some embodiments, the RNA polynucleotides of the norovirus vaccine encode antigenic polypeptides from four different norovirus genotypes, i.e., the vaccine is "tetravalent." In certain embodiments, the RNA polynucleotides of the norovirus vaccine encode antigenic polypeptides from five different norovirus genotypes, i.e., the vaccine is "pentavalent."

[0080] In some embodiments, the polynucleotides of the present disclosure are codon-optimized. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match the codon frequency in the target organism and the host organism to ensure proper folding; bias the GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove, or shuffle protein domains; insert or modify restriction sites, 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 include services from 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.

[0081] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity, less than 90% sequence identity, less than 85% sequence identity, less than 80% sequence identity, or less than 75% sequence identity with a naturally occurring or wild-type sequence. In certain embodiments of the invention, the codon-optimized polynucleotide sequence is any one of SEQ ID NOs: 1-5 or 9-13, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments of the invention, the codon-optimized polynucleotide sequence is any one of SEQ ID NOs: 1-5 or 9-13.

[0082] In addition to the coding region, mature mRNA contains a 5' untranslated region (UTR) and a 3' UTR, which play important roles in regulating gene expression. "5' UTR" refers to a region of an mRNA that does not encode a polypeptide and is located immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript that is translated by a ribosome). "3' UTR" refers to a region of an mRNA that does not encode a polypeptide and is located immediately downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals the end of translation). In some embodiments of the present invention, the 5' UTR is SEQ ID NO: 6. In some embodiments, the 3' UTR is SEQ ID NO: 7 or SEQ ID NO: 8.

[0083] The 5' UTR may further comprise a 5' cap sequence. 5'-capping of polynucleotides may be completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5' guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, Massachusetts) according to the manufacturer's protocol. 5'-capping of modified RNAs may be completed post-transcriptionally using vaccinia virus capping enzyme to generate the "Cap0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Massachusetts). To generate m7G(5')ppp(5')G-2'-O-methyl, a Cap1 structure can be generated using both vaccinia virus capping enzyme and 2'-O-methyl-transferase. A Cap2 structure can be generated from the Cap1 structure, followed by 2'-O-methylation of the third-to-last 5' nucleotide using a 2'-O-methyl-transferase. A Cap3 structure can be generated from the Cap2 structure, followed by 2'-O-methylation of the fourth-to-last 5' nucleotide using a 2'-O-methyl-transferase. The enzymes are preferably derived from recombinant sources. In certain embodiments of the invention, the 5' cap structure is N7-methyl-G(3'OMe)ppp.

[0084] The 3' UTR may further comprise a poly(A) sequence or a poly(C) sequence, which is typically located at the 3' end of the mRNA. The poly(A) sequence, also referred to as a poly(A) tail or 3' poly(A) tail, is typically understood to be a sequence of adenosine nucleotides, for example, up to about 400 adenosine nucleotides (SEQ ID NO: 29), for example, about 10 to about 400, preferably about 10 to about 200, more preferably about 20 to about 80, even more preferably about 40 to about 80, and most preferably about 60 to about 80 adenosine nucleotides. In a specific embodiment of the present invention, the poly(A) sequence comprises 80 adenine nucleotides (SEQ ID NO: 24). A poly(C) sequence, also referred to as a poly(C) tail or 3' poly(C) tail, is typically understood to be a sequence of cytidine nucleotides, for example, up to about 400 cytidine nucleotides (SEQ ID NO: 31), for example, about 10 to about 400 cytidine nucleotides, preferably about 10 to about 200 cytidine nucleotides, more preferably about 20 to about 80 cytidine nucleotides, even more preferably about 40 to about 80 cytidine nucleotides, and most preferably about 60 to about 80 cytidine nucleotides. In a specific embodiment of the present invention, the poly(C) sequence comprises 80 cytidine nucleotides (SEQ ID NO: 30).

[0085] In the context of the present invention, poly(A) sequences can be located within mRNA or any other nucleic acid molecule, for example, in a vector, such as in a vector that serves as a template for the production of RNA, preferably mRNA, by transcription of the vector. Furthermore, poly(A) sequences or poly(A) tails can be generated in vitro by enzymatic polyadenylation of RNA, for example, using poly(A) polymerase (PAP) derived from Escherichia coli (E. coli) or yeast. Furthermore, polyadenylation of RNA can be achieved, for example, by using immobilized PAP enzyme in a polyadenylation reactor. See International Patent Application Publication No. 2016 / 174271.

[0086] chemical modification The RNA vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one Norovirus antigenic polypeptide comprising at least one chemical modification.

[0087] The terms "chemical modification" and "chemically modified" refer to modifications to adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides in at least one of the following: position, pattern, percentage, or population of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides. Generally, these terms do not refer to naturally occurring ribonucleotide modifications in the 5'-terminal mRNA cap portion. With respect to polypeptides, the term "modification" refers to modifications to the standard set of 20 amino acids. The polypeptides provided herein are also considered "modified" if they contain amino acid substitutions, insertions, or a combination of substitutions and insertions.

[0088] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises a variety (more than one) different modifications. In some embodiments, a specific region of a polynucleotide contains one, two, or more (which may be different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or organism exhibits reduced degradation in the cell or organism, respectively, compared to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., a reduced innate response) in the cell or organism, respectively.

[0089] Modifications of polynucleotides include, but are not limited to, those described herein.Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) can contain naturally occurring modifications, non-naturally occurring modifications, or polynucleotides can contain a combination of naturally occurring modifications and non-naturally occurring modifications.Polynucleotides can contain any useful modifications, for example, of sugars, nucleobases, or internucleoside linkages (e.g., to the phosphate linkage, to the phosphodiester bond, or to the phosphodiester backbone).

[0090] Polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides) in some embodiments contain non-naturally occurring modified nucleotides that are introduced during or after the synthesis of the polynucleotide to achieve a desired function or property. Modifications can be on the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced at the end of the chain or anywhere else in the chain, using chemical synthesis or using polymerase enzymes. Any region of a polynucleotide can be chemically modified.

[0091] The present disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA polynucleotides such as mRNA polynucleotides). A "nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or its derivative in combination with an organic base (e.g., a purine or pyrimidine) or its derivative (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method, e.g., chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A polynucleotide can contain one or more regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide would contain a region of nucleotides.

[0092] 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 containing non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of the present disclosure.

[0093] Modifications of polynucleotides (e.g., RNA polynucleotides such as 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,N6-Dimethyladenosine;N6-Acetyladenosine;N6-Hydroxynor Valylcarbamoyladenosine;N6-Methyl-N6-threonylcarbamoyladenosine;2-Methyladenosine;2-Methylthio-N6-isopentenyladenosine;7-Deazaadenosine;N1-Methyladenosine;N6,N6(dimethyl)adenine;N6-cis-Hydroxy-isopentenyladenosine;α-Thioadenosine;2(Amino)adenine;2(Aminopropyl)adenine;2(Methylthio)N6(isopentenyl)adenine;2-(Alkyl)adenines;2-(Aminoalkyl)adenines )adenine;2-(aminopropyl)adenine;2-(halo)adenine;2-(halo)adenine;2-(propyl)adenine;2'-amino-2'-deoxy-ATP;2'-azido-2'-deoxy-ATP;2'-deoxy-2'-α-aminoadenosine TP;2'-deoxy-2'-α-azidoadenosine TP;6(alkyl)adenine;6(methyl)adenine;6-(alkyl)adenine;6-(methyl)adenine;7(deaza)adenine;8(alkenyl)adenine;8(alkynyl)adenine;8(Amino)adenine;8(Thioalkyl)adenine;8-(Alkenyl)adenine;8-(Alkyl)adenine;8-(Alkynyl)adenine;8-(Amino)adenine;8-(Halo)adenine;8-(Hydroxyl)adenine;8-(Thioalkyl)adenine;8-(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'-a-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 T P;2-Mercaptoadenosine TP;2-Methoxy-adenine;2-Methylthio-adenine;2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP;3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP;3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP;4'-Azidoadenosine TP;4'-Carbocyclic adenosine TP;4'-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; Lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-Aza-cytidine; Pseudo-isocytidine; 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)cytidine 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-Bromocytidine;5-Iodocytidine;5-Propynylcytosine;6-(Azo)cytosine;6-Azacytidine N4(acetyl)cytosine;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 Cytidine TP; 2'-deoxy-2'-a-mercaptocytidine 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 N4-amino-cytidine TP; N4-benzoyl-cytidine 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-thioguanosine;7-deazaguanosine;8-oxoguanosine;N1-methylguanosine;α-thioguanosine;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-(Hydroxy)guanine (hydroxyl)guanine;8-(thioalkyl)guanine;8-(thiol)guanine;Azaguanine;Deazaguanine;N(methyl)guanine;N-(methyl)guanine;1-Methyl-6-thioguanosine;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-thioguanosine;1-Me-GTP;2'-Fluoro 2'-O-methyl-N2-isobutyl-guanosine TP; 2'-α-ethynylguanosine TP; 2'-α-trifluoromethylguanosine TP; 2'-β-ethynylguanosine TP; 2'-β-trifluoromethylguanosine TP; 2'-deoxy-2',2'-difluoroguanosine TP; 2'-deoxy-2'-α-mercaptoguanosine TP; 2'-deoxy-2'-α-thiomethoxyguanosine TP; 2'-deoxy-2'-β-aminoguanosine TP; 2'-deoxy-2'-β-azidoguanosine TP;2'-Deoxy-2'-b-bromoguanosine TP; 2'-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 TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5'-Homoguanosine TP; 8-Bromoguanosine TP; 9-Deazaguanosine TP; N2-Isobutylguanosine TP; 1-Methylinosine; Inosine; 1,2'-O-Dimethylinosine; 2'-O-Methylinosine; 7-Methylinosine; 2'-O-Methylinosine; Epoxyqueuosine; Galactosylqueuosine; Mannosylqueuosine; Queuosine; Allylamino-thymidine; Azathymidine; Deazathymidine; Deoxythymidine; 2'-O-methyluridine;2-thiouridine;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-methylpseudo(pseduo)uridine;1-methyl-pseudouridine;2'-O-methyluridine;2'-O-methylpseudouridine; 2'-O-Methyluridine;2-Thio-2'-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine;3,2'-O-Dimethyluridine;3-Methyl-pseudouridine TP;4-Thiouridine;5-(Carboxyhydroxymethyl)uridine;5-(Carboxyhydroxymethyl)uridine methyl ester;5,2'-O-Dimethyluridine;5,6-Dihydrouridine;5-Aminomethyl-2-thiouridine;5-Carbamoylmethyl-2'-O-methyluridine;5-Carbamoylmethyluridine;5-Carboxyhydro hydroxymethyluridine;5-carboxyhydroxymethyluridine 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;Uridine 5-hydroxyacetic acid;Uridine 5-hydroxyacetic acid methyl ester;3-(3-amino-3-carboxypropyl)-uridine TP;5-(isopentenylaminomethyl)-2-thiouridine TP;5-(iso -pentenylaminomethyl)-2'-O-methyluridine TP;5-(iso-pentenylaminomethyl)uridine TP;5-propynyluracil;α-thio-uridine;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'fluro-guanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2'deoxyuridine;2'-Fluorouridine;2'-Deoxy-2'-α-aminouridine TP;2'-Deoxy-2'-α-azidouridine TP;2-Methylpseudouridine;3(3amino-3carboxypropyl)uracil;4(thio)pseudouracil;4-(thio)pseudouracil;4-(thio)uracil;4-Thiouracil;5(1,3-Diazol-1-alkyl)uracil;5(2-aminopropyl)uracil;5(Aminoalkyl)uracil;5(Dimethylaminoalkyl)uracil;5(Guanidinium alkyl)uracil, 5(Methoxycarbonyl 5-(methylaminomethyl)-2-(thio)uracil;5-(methoxycarbonylmethyl)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)uracil )pseudouracil;5-(Alkyl)-4(thio)pseudouracil;5-(Alkyl)pseudouracil;5-(Alkyl)uracil;5-(Alkynyl)uracil;5-(Allylamino)uracil;5-(Cyanoalkyl)uracil;5-(Dialkylaminoalkyl)uracil;5-(Dimethylaminoalkyl)uracil;5-(Guanidiniumalkyl)uracil;5-(Halo)uracil;5-(l,3-Diazole-l-alkyl)uracil;5-(Methoxy)uracil;5-(Methoxycarbonylmethyl)-2-(thio)uracil;5-( 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)URACIL5-(Trifluoromethyl)uracil;5-Aminoallyl-uridine;5-Bromo-uridine;5-Iodouridine;5-Uracil;6(Azo)uracil;6-(Azo)uracil;6-Aza-uridine;Alliamino(allyamino)-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-hydroxypropyl)pseudouridine TP;(2R)-1-(2-hydroxypropyl)pseudouridine TP;(2S)-1-(2-hydroxypropyl)pseudouridine TP;(E)-5-(2-bromo-vinyl)ara-uridine TP;(E)-5-(2-bromo-vinyl)uridine TP;(Z)-5-(2-bromo-vinyl)ara-uridine TP;(Z)-5-(2-bromo-vinyl)uridine TP;1-(2,2,2-trifluoroethyl)-pseudo-UTP;1- (2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2-diethoxyethyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudo-UTP; 1-(2,4,6-trimethylphenyl)pseudo-UTP; 1-(2-amino-2-carboxyethyl)pseudo-UTP; 1-(2-amino-ethyl)pseudo-UTP; 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-UT P;1-(4-Azidobenzyl)pseudouridine TP;1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP;1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP;1-(4-Methoxyphenyl)pseudouridine TP;1-(4-Methylbenzyl)pseudouridine T P;1-(4-Methyl-benzyl)pseudo-UTP;1-(4-Nitrobenzyl)pseudo-UTP;1-(4-Nitro-benzyl)pseudo-UTP;1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP;1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1 -[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine TP; 1-acetylpseudouridine TP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-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-pseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudouridine TP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Bu; 1-Cyanomethylpseudo-UTP;1-Cyanomethylpseudo-uridine 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-pseudo-UTP; 1-hexyl-pseudo-UTP; 1-homoallylpseudouridine TP; 1-hydroxymethylpseudouridine TP; 1-isopropyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-α-thio-pseudo-UTP; 1-methanesulfonylmethylpseudouridine TP; 1-methoxymethylpseudouridine TP; 1-methyl-6-(2,2,2-trifluoroethyl)pseudouridine TP -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-ethyano-pseudo-UTP Toxo-pseudo-UTP; 1-methyl-6-ethylcarboxylate-pseudo-UTP; 1-methyl-6-ethyl-pseudo-UTP; 1-methyl-6-fluoro-pseudo-UTP; 1-methyl-6-formyl-pseudo-UTP; 1-methyl-6-hydroxyamino-pseudo-UTP; 1-methyl-6-hydroxy-pseudo-UTP; 1-methyl-6-iodo-pseudo-UTP; 1-methyl-6-isopropyl-pseudo-UTP; 1-methyl-6-methoxy-pseudo-UTP; 1-methyl-6-methylamino-pseudo-UTP;1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP;1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP;1-Morpholinomethylpseudouridine TP;1-Pentyl-pseudo-UTP;1-Phenyl-pseudo-UTP;1-Pivaloylpseudouridine TP;1-Propargylpseudouridine TP;1-Propyl-pseudo-UTP;1-Propynyl-pseudouridine;1 -p-Tolyl-pseudo-UTP; 1-tert-butyl-pseudo-UTP; 1-thiomethoxymethylpseudouridine TP; 1-thiomorpholinomethylpseudouridine TP; 1-trifluoroacetylpseudouridine TP; 1-trifluoromethyl-pseudouridine TP; 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' -a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy-2'-a-mercaptopyridine 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-iodouridine TP; 2'-Deoxy-2'-b-mercaptolysine 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 -UTP;6-Butyl-pseudo-UTP;6-Chloropseudo-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;α-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)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(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 esterPseudo-UTP-N1-3-propionic acid; Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid; Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid; Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; Undermodified hydroxywybutosine; 4-Demethylwybutosine 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)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-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-yl;7-(Guanidinium alkylhydroxy)-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)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl (hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Propynyl)isocarbostyrilyl;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 Bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Difluorotolyl;Hypoxanthine;Imidizopyridinyl;Inosinyl;Isocarbostyrilyl;Isoguanidine;N2-Substituted Purines;N6-Methyl-2-amino-purine;N6-Substituted Purines;N-Alkylated Derivatives;Napthalenyl;Nitrobenzimidazolyl;Nitroimidazolyl;Nitroindazolyl;Nitropyrazolyl;Nubularine;O6-Substituted Purines;O-Alkylated Derivatives;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; Propynyl-7-(aza; )Indolyl;Pyrenyl;Pyridopyrimidin-3-yl;Pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl;Pyrrolopyrimidin-2-one-3-yl;Pyrrolopyrimidinyl;Pyrrolopyridinyl;Stilbenzyl;Substituted 1,2,4 triazoles;Tetracenyl;Tubercidin;Xanthine;Xanthosine-5'-TP;2-Thio-Zebularine;5-Aza-2-Thio-Zebularine;7-Deaza-2-amino-Purine;Pyridin-4-one ribonucleoside;2-Amino-riboside-TP;Formycin A TP;Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0094] In some embodiments, a polynucleotide (eg, an RNA polynucleotide such as an mRNA polynucleotide) comprises a combination of at least two (eg, two, three, four, or more) of the above modified nucleobases.

[0095] 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), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In some embodiments, 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.

[0096] In some embodiments, one or more polynucleotides of the present invention are modified by replacing one or more uridine residues with one or more modified nucleobases. In certain embodiments, at least 50% of the uridine residues of an mRNA polynucleotide are replaced with N1-methylpseudouridine. In certain embodiments, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% of the uridine residues of an mRNA polynucleotide are replaced with N1-methylpseudouridine. In certain embodiments, 90% of the uridine residues of an mRNA polynucleotide are replaced with N1-methylpseudouridine. In certain embodiments, 91% of the uridine residues of an mRNA polynucleotide are replaced with N1-methylpseudouridine. In certain embodiments, 92% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridines. In certain embodiments, 93% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridines. In certain embodiments, 94% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridines. In certain embodiments, 95% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridines. In certain embodiments, 96% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridines. In certain embodiments, 97% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridines. In certain embodiments, 98% of the uridine residues of the mRNA polynucleotide are replaced with N1-methylpsuedouridine.In certain embodiments, 99% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpsuedouridine, hi certain embodiments, 100% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpsuedouridine.

[0097] 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 In some embodiments, the polynucleotide comprises a combination of at least two (e.g., two, three, four, or more) of the above modified nucleobases.

[0098] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (mC). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (mψ). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (mψ) and 5-methyl-cytidine (mC). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (mC). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0099] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide such as an mRNA polynucleotide) is uniformly modified (e.g., fully modified, modified throughout the sequence) with respect to a particular modification. For example, the polynucleotide may contain 5-methyl-cytidine (m 5 C), and all cytosine residues in the mRNA sequence are uniformly modified with 5-methyl-cytidine (m 5 C). Similarly, polynucleotides may be uniformly modified with respect to any type of nucleoside residue present in the sequence by replacement with a modified residue such as those described above.

[0100] 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.

[0101] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified cytosine or modified uridine include 5-cyanouridine and 4'-thiouridine.

[0102] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0103] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0104] The polynucleotide of the present disclosure can be partially or completely modified along the entire length of the molecule.For example, one or more, or all, or a given type of nucleotide (for example, purine or pyrimidine, or any one or more or all of A, G, U, C) can be uniformly modified in the polynucleotide of the present disclosure or in a given predetermined sequence region (for example, in mRNA, including or excluding poly(A) tail).In some embodiments, all nucleotides X in the polynucleotide of the present disclosure (or in a given sequence region) are modified nucleotides, and X can 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.

[0105] Polynucleotides may contain (with respect to overall nucleotide content, or with respect to any one or more types of nucleotides, i.e., A, G, U, or C) from about 1% to about 100% modified nucleotides, or any percentage therebetween (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-95%, 10%-10 ... % to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It will be understood that any remaining percentage is accounted for by unmodified A, G, U or C occurrences.

[0106] A polynucleotide may contain at least 1% and at most 100% modified nucleotides, or any percentage therebetween, such as 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 are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils can be replaced by a compound with a single unique structure, or by multiple compounds with different structures (e.g., 2, 3, 4, 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 the polynucleotide are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be replaced by a compound having a single unique structure, or by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0107] In vitro transcription (IVT) of RNA (e.g., mRNA) "In vitro transcription (IVT)" is a method of synthesizing mRNA from a linear DNA template using an RNA polymerase, which binds to a promoter sequence and transcribes the mRNA by adding complementary nucleotides in the 5' to 3' direction. The norovirus vaccines of the present disclosure include at least one RNA polynucleotide, such as an mRNA (e.g., a modified mRNA). The mRNA is transcribed in vitro, for example, from a template DNA called an "in vitro transcription template." In some embodiments, the at least one RNA polynucleotide has at least one chemical modification. The at least one chemical modification may include, but is not expressly limited to, any of the modifications described herein.

[0108] A linear DNA template encoding an mRNA sequence is prepared from purified plasmid DNA and may contain an RNA polymerase promoter sequence, an encoded 5' UTR, a coding sequence, an encoded 3' UTR with a poly(A) tail, and a linearization cleavage site. The plasmid DNA sequence may be pUC19, pUC57, or pmRNA. XPThe plasmid DNA is generated using standard cloning methods in a plasmid vector backbone such as , and the plasmid DNA is purified from fermentation of a transformed E. coli strain typically used for plasmid production, such as Stable or DH5α, using standard methods (see, e.g., U.S. Patent Application Publication No. 2019 / 0083602 and U.S. Patent Application Publication No. 2020 / 0392518). mRNA can be produced with a co-transcribed cap analog adduct and a poly(A) tail encoded by the DNA template in the IVT reaction, or the cap and / or poly(A) tail can be enzymatically added after the IVT reaction. The DNA plasmid can contain an adenine-guanine (AG) sequence at the beginning of the 5'UTR after the T7 promoter sequence to enable capping of the co-transcription with the Cap1AG analog (m7G(5')ppp(5')(2'OMeA)pG). The DNA plasmid can also contain a specific restriction enzyme cleavage site after the encoded 3' poly(A) tail. Plasmids can be linearized in a digestion reaction containing purified plasmid DNA at a concentration of about 0.1-2 mg / mL and an appropriate restriction enzyme, such as BbsI or BspQI, at a concentration of about 500-20,000 U / mg, in a digestion mixture that may contain Tris-HCl, magnesium, potassium, acetate, egg white, and / or other excipients, at a solution pH of about 7-8. The reaction is incubated at a temperature range of about 34-40°C, preferably 37°C, for about 30-90 minutes, preferably 60 minutes.

[0109] The linear DNA template can be purified by solvent extraction, alcohol precipitation, centrifugation, chromatography, and / or filtration-based methods to remove uncut plasmid DNA, restriction enzyme, and digested mixture components. The purified linear DNA template can be prepared at a concentration of approximately 0.5-2 mg / mL, preferably 1 mg / mL, in water or a buffer containing Tris-HCl, ethylenediaminetetraacetic acid (EDTA), and / or other excipients at a solution pH of approximately 7-8.

[0110] The IVT reaction is carried out in a temperature-controlled reaction vessel with an incubation temperature range of approximately 34-40°C, preferably 37°C. The linear DNA template may be added to the IVT reaction mixture at a concentration range of approximately 10-200 μg / mL, preferably 40-60 μg / mL. Natural nucleoside triphosphates (NTPs), including adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), or pseudouridine (Ψ), N-terminal nucleotides, may be added. 1 -Methylpseudouridine (m 1 Ψ), N 6 -methyladenosine (m 6 A) or N 5 -methylcytidine (m 5 C) modified NTPs such as ATP, GTP, CTP and m 1 Ψ can be used in IVT reactions. NTP is 50% UTP and 50% m 1 Mixtures of natural and substituted modified NTPs can be incorporated into mRNA, such as a mixture of Ψ. Preferably, 100% m is used to completely replace UTP. 1 Ψ is added. NTPs are added at about 2 to 15 mM, or preferably 8 to 14 mM for ATP, GTP, and CTP. 1 Ψ can be added to the IVT reaction mixture at a concentration ranging from 4 to 7 mM. A 5'Cap 1 AG analog, such as CleanCap AG(3'OMe)(m7(3'OMeG)(5')ppp(5')(2'OMeA)pG) or CleanCap AG(m7(5')ppp(5')(2'OMeA)pG), preferably CleanCap AG(3'OMe), can be added to the IVT reaction mixture at a concentration ranging from approximately 1 to 10 mM, preferably 3 to 5 mM. RNA polymerase, such as T7 RNA polymerase, can be added to the IVT reaction mixture at a concentration ranging from approximately 2,000 to 20,000 U / mL, preferably 8,000 to 12,000 U / mL.

[0111] Pyrophosphatases, such as yeast inorganic pyrophosphatase, and RNase inhibitors, such as mouse RNase inhibitor, can be added to the IVT reaction mixture at concentrations ranging from 1 to 5 U / mL and 500 to 2,000 U / mL, respectively. Other components of the IVT reaction mixture may include Tris-HCl, magnesium, spermidine, dithiothreitol (DTT), sodium chloride, potassium acetate, phosphate, polysorbate-20, polysorbate-80, Triton X-100, glycerol, and / or other excipients, with a final solution pH of approximately 6 to 9. The IVT reaction mixture is typically incubated with mixing for 1 to 6 hours, preferably 3 to 4 hours. Preferably, mRNA is produced in the IVT reaction with a cotranscribed 5' Cap 1 AG analog and a 3' poly(A) tail encoded by the DNA template, eliminating the need for additional enzymatic capping or poly(A) tailing. The IVT reaction is terminated by the addition of a DNase, such as DNase I, to digest the linear DNA template into small oligonucleotides and terminate transcription. DNase is added to the IVT reaction mixture at a concentration range of approximately 100-500 U / mL, preferably 350 U / mL, along with calcium chloride at a concentration range of approximately 1-5 mM, preferably 3.5 mM, and incubated at approximately 34-40°C, preferably 37°C, for 0.5-3 hours, preferably 1 hour. After DNase digestion, a proteinase, such as proteinase K, can be added to the IVT reaction mixture to digest the enzymes used in the IVT reaction into small peptides. The proteinase is added to the IVT reaction mixture at a concentration range of approximately 0.05-0.2 mg / mL, preferably 0.1 mg / mL, along with sodium dodecyl sulfate (SDS) and additional DTT, and incubated at approximately 34-40°C, preferably 37°C, for 0.5-3 hours, preferably 1 hour. Preferably, only DNase treatment is performed, without proteinase treatment. After the DNase or DNase followed by proteinase treatment is completed, the IVT reaction mixture is adjusted to 10-100 mM EDTA, preferably 50 mM, to quench the enzyme activity.

[0112] The resulting IVT reaction mixture contains the target mRNA sample and contaminants, such as template DNA and digested oligonucleotides, enzymes including RNA polymerase, pyrophosphatase, RNase inhibitor, DNase and proteinase, small molecules including free nucleotides, pyrophosphate, magnesium, spermidine and / or other IVT reaction matrix excipients, and RNA-related contaminants such as RNA fragments, double-stranded RNA, uncapped RNA, or RNA lacking a poly(A) tail.

[0113] An initial tangential flow filtration (TFF) step may be performed after DNase and proteinase treatment for buffer exchange and removal of small impurities. TFF is performed by pumping the feed solution across a membrane at a transmembrane pressure (TMP) of approximately 1-10 psi; solution components larger than the molecular weight cut-off (MWCO) remain in the retentate, while components smaller than the MWCO permeate through the membrane. Membrane MWCOs can range from 30-500 kDa in flat-sheet or hollow-fiber formats and can be composed of a variety of materials, including polyethersulfone (PES), polysulfone (PS), or regenerated cellulose (RC). As the IVT reaction mixture is processed across TFF, large mRNA molecules are retained, while digested impurities and other small molecules are removed in the permeate. Before initiating TFF, the IVT reaction mixture may be diluted 2-20-fold in water or a buffer containing Tris-HCl, sodium phosphate, or sodium citrate at a concentration of about 1-100 mM, preferably 10 mM Tris-HCl, at a pH of about 6-8, and EDTA at a concentration of about 1-10 mM, preferably 2 mM. To buffer exchange the diluted IVT mixture across 5-15 diafiltration volumes (DV) into water or a buffer containing Tris-HCl, sodium phosphate, or sodium citrate at a concentration of about 1-100 mM, at a pH of about 6-8, and EDTA at a concentration of about 1-10 mM, use a TMP of about 3-7 psi and a flow rate of about 2,000-12,000 s. -1 Approximately 1-25 g of mRNA / m2 operated at cross-flow shear rates of 2A TFF membrane loaded with the diluted IVT reaction mixture can be used. Preferably, a TFF membrane with approximately 4 psi of TMP and 4000 s is used to buffer exchange the diluted IVT mixture into 10 mM Tris-HCl, 2 mM EDTA, pH 7.2 across 10 DV. -1 5-10 g mRNA / m operated at cross-flow shear rates of 2 A 50 kDa hollow fiber PES membrane loaded with PEG is used.

[0114] The purified mRNA-containing solution in the TFF retentate can be applied to a hybridization affinity chromatography medium that uses oligodeoxythymine (oligo-dT) ligands conjugated to a stationary phase to selectively bind mRNA through complementary base pairing between the oligo-dT ligand and the mRNA poly(A) tail. IVT reaction contaminants, including RNA polymerase, pyrophosphatase, RNase inhibitors, DNase, and proteinases, as well as small molecules including free nucleotides, pyrophosphate, magnesium, spermidine, other IVT matrix excipients, and RNA fragments lacking poly(A) tails, are not expected to bind to the oligo-dT ligand. Preferably, the DNase-treated IVT reaction mixture is applied directly to oligo-dT affinity chromatography, omitting the proteinase K and initial TFF steps. The oligo-dT ligand can consist of approximately 15-30 deoxythymine bases connected to a linker that couples the ligand to a support surface such as a microporous polymethacrylate monolith, cross-linked poly(styrene-divinylbenzene) beads, or electrospun cellulose nanofibers. The mRNA-containing solution sent to the oligo-dT hybridization affinity chromatography step can be diluted approximately 2-50-fold into an oligo-dT binding matrix consisting of approximately 100-1000 mM salt, such as sodium chloride, potassium chloride, lithium chloride, guanidine hydrochloride, or other similar salts; a buffer solution, such as Tris-HCl, sodium phosphate, or sodium citrate, at a pH of approximately 6-8, at a concentration of 5-100 mM; and approximately 1-10 mM EDTA. Preferably, the DNase-treated IVT mixture is diluted approximately 12-fold into an oligo-dT binding matrix consisting of 400 mM sodium chloride, 10 mM Tris-HCl, 2 mM EDTA, pH 7.2.The mRNA-containing solution can be pumped through oligo(dT) chromatography media at an approximate loading of about 1-8 mg / mL medium with a residence time of about 0.1-10 minutes. Once the poly(A)-tailed mRNA binds, the column is washed with 2-10 column volumes (CV) of mobile phase consisting of a salt, such as sodium chloride, potassium chloride, lithium chloride, guanidine hydrochloride, or other similar salt, at a concentration of about 10-200 mM, a buffer, such as Tris-HCl, sodium phosphate, or sodium citrate, at a concentration of 5-100 mM, and EDTA at a concentration of about 1-10 mM. Preferably, the mRNA-containing mixture is pumped through a microporous polymethacrylate monolith media at a loading of 2-3 mg / mL medium and a residence time of 0.5 minutes. The column is washed with 5 CV of a buffer consisting of 50 mM sodium chloride, 10 mM Tris-HCl, 2 mM EDTA, pH 7.2. The bound mRNA is eluted from the oligo dT ligand using 2-10 CV of a low ionic strength mobile phase consisting of water or a buffer such as Tris-HCl, sodium phosphate, or sodium citrate at a concentration of 1-20 mM, at a pH of approximately 6-8. Preferably, the bound mRNA is eluted with 4-5 CV of 10 mM Tris-HCl, pH 7.2. The oligo dT chromatography medium can be regenerated with sodium hydroxide and reused for subsequent chromatography purification cycles. Preferably, the polymethacrylate monolith is regenerated with 0.5 M sodium hydroxide and reused through approximately 2-6 purification cycles of the same DNase-treated IVT mixture diluted in oligo dT binding buffer.

[0115] The mRNA-containing oligo dT hybridization affinity chromatography elution fraction can be purified by a final purification chromatographic method to remove residual RNA-related impurities such as RNA fragments, double-stranded mRNA, and uncapped RNA, or residual enzymes such as RNA polymerase. Examples of final purification chromatographic methods include mixed-mode chromatography, hydrophobic interaction chromatography, anion exchange chromatography, reversed-phase chromatography, ceramic hydroxyapatite chromatography, size exclusion chromatography, or cellulose chromatography. Preferably, no further final purification method is performed after oligo dT chromatography.

[0116] The mRNA-containing oligo dT hybridization affinity chromatography elution fraction is buffer exchanged and purified by a final TFF, which can be performed similarly to the initial TFF described above. The final TFF membrane can be operated at 1-10 psi TMP using a 30-500 kDa MWCO membrane in flat sheet or hollow fiber format and can be composed of polyethersulfone (PES), polysulfone (PS), or regenerated cellulose (RC). The oligo dT elution fraction is concentrated approximately 2-20 times, followed by buffer exchange across 4-15 DV into a buffer consisting of water or approximately 1-10 mM Tris-HCl, sodium phosphate, or sodium citrate at a pH of approximately 5-8, at 3-7 psi TMP and approximately 2,000-12,000 s. -1 Operated at cross-flow shear rates of approximately 1-20 g mRNA / m 2 A 50-100 kDa hollow fiber PES membrane loaded with a membrane area of ​​100 kDa can be used. Preferably, for an 8-fold concentration followed by a buffer exchange of 6 DV into 1 mM sodium citrate, pH 6.4, a TMP of approximately 4 psi and 4000 s -1 5-10 g mRNA / m operated at cross-flow shear rates of 2 A 50 kDa hollow fiber PES membrane loaded with PEG is used.

[0117] The final mRNA-containing retentate from TFF was approximately 25–1000 L / m 2 -hr flux and approximately 10-1000g / m 2 The final TFF retentate is filtered through a bioburden reduction filter having a nominal pore size of about 0.2 μm at a loading of 100 μL / ml. The filter may be constructed from a variety of materials, including hydrophilic polyvinylidene fluoride (PVDF), PES, or cellulose acetate, and may contain a prefilter having a nominal pore size ranging from about 0.2 to 1 μm. Preferably, the final TFF retentate is filtered through a 0.2 μm PVDF filter at a flow rate of 300 L / ml. 2 -hr flux and 100g / m 2 The bioburden-reduced filter product is pumped at a loading of 1000 bp. The concentration of mRNA in the bioburden-reduced filter product is calculated from sample measurements of absorbance at 260 nm using a spectrophotometer. The filtered product may be diluted with final TFF diafiltration buffer to a target final concentration of mRNA ranging from about 0.5 to 5 mg / mL. Preferably, the filtered product is diluted with 1 mM sodium citrate, pH 6.4, to an mRNA concentration of about 1 mg / mL. The purified mRNA may be stored under refrigerated conditions at about 2 to 8°C, or frozen at about -20°C or below -60°C.

[0118] antigenic polypeptides An "antigenic polypeptide" is a polypeptide that induces an immune response when administered to an animal, preferably a mammal. In some embodiments, antigenic polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. In certain embodiments, the antigenic polypeptide is a norovirus VP1 or VP2 polypeptide. In certain embodiments, the antigenic polypeptide is any one of SEQ ID NOS: 14-18. The antigenic polypeptides of the present disclosure can be prepared by any suitable method known in the art, including synthetic methods such as solid-phase synthesis, and recombinant and in vitro methods such as in vitro transcription reactions. In certain embodiments, the antigenic polypeptides can be prepared by in vitro transcription of the polynucleotides disclosed herein.

[0119] A "variant" is a molecule whose amino acid or nucleic acid sequence differs from a native or reference sequence. A sequence variant may have any two or three substitutions, deletions, insertions, or combinations of the foregoing at a position within the amino acid or nucleic acid sequence compared to the native or reference sequence. Typically, a variant has at least 50% identity to the native or reference sequence. In some embodiments, a variant shares at least 80% identity or at least 90% identity with the native or reference sequence.

[0120] 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 synonymous with the term "variant" and generally refers to a molecule that is modified and / or changed in some way compared 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 the polypeptide sequences disclosed herein, are within the scope of the present disclosure.

[0121] A "substitution variant" when referring to a polynucleotide is a variant in which at least one nucleotide residue in the native or starting sequence has been removed and a different nucleotide has been inserted in its place at the same position. In some embodiments, one or more uridine residues in the mRNA polynucleotide of the present disclosure are replaced with pseudouridine. For example, the modification of mRNA nucleosides with pseudouridine or pseudouridine derivatives has been previously described. See International Patent Application Publication No. 2007 / 024708.

[0122] As used herein, the terms "terminus" or "end" when referring to a polypeptide or polynucleotide refer to the end of the polypeptide or polynucleotide, respectively. Such ends are not limited to the first or last position of the polypeptide or polynucleotide, but may include additional amino acids or nucleotides in the terminal region.

[0123] 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, with 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 sequences, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if present), handled 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, if necessary, introducing gaps to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. The identity depends on the calculation of percent identity, but values ​​may vary due to gaps and penalties introduced into the calculation. Generally, a variant of a particular polynucleotide or polypeptide will have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity with that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those in the BLAST suite.See 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. See Smith, TF & Waterman, MS (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. See Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins," J. Mol. Biol. 48:443-453. More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0124] Lipid nanoparticles (LNPs) As used herein, "lipid nanoparticle" or "LNP" refers to any lipid composition that can be used to deliver a product, including, but not limited to, a liposome or vesicle, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; monolayer or multiple; multilayer), or in other embodiments, the lipid coats an interior containing a prophylactic or therapeutic product, i.e., a lipid aggregate or micelle, in which the lipid-encapsulated prophylactic or therapeutic product is contained within a relatively disordered lipid mixture. In certain embodiments, mRNA polynucleotides of the invention can be co-encapsulated in LNPs that contain one or more cationic or polycationic compounds. Except as otherwise noted, LNPs need not incorporate an antigenic polypeptide therein, and can be used to deliver a product (i.e., an mRNA polynucleotide) to a mammal when the LNP and mRNA polynucleotide are in the same formulation.

[0125] As used herein, "polyamine" means a compound with two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.

[0126] Unless otherwise specified, mole % refers to mole percent of total lipids.

[0127] Generally, the LNPs of the compositions of the present invention are composed of one or more cationic lipids (including ionizable cationic lipids) and one or more poly(ethylene glycol)-lipids (PEG-lipids). In certain embodiments, the LNPs further comprise one or more non-cationic lipids. The one or more non-cationic lipids can comprise phospholipids, phospholipid derivatives, sterols, fatty acids, or combinations thereof.

[0128] Cationic lipids and ionizable cationic lipids suitable for LNPs are described herein. Ionizable cationic lipids are characterized by the weak basicity of their lipid head groups, which affects the surface charge of the lipid in a pH-dependent manner, making them positively charged at acidic pH but close to charge neutral at physiological pH. Cationic lipids are characterized by monovalent or polyvalent cationic charges on their head groups, which make them positively charged at neutral pH. In certain embodiments, cationic and ionizable lipids can form complexes with hydrophilic bioactive molecules to produce hydrophobic complexes that partition into the organic phase of a two-phase aqueous / organic system. It is believed that both monovalent and polyvalent cationic lipids can be used to form hydrophobic complexes with bioactive molecules.

[0129] Preferred cationic and ionizable cationic lipids used to form LNPs include N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3 dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3 dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride. ("DODAP"); 1,2 bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP); 3-(N-(N,N-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"); diheptadecylamidoglycylspermidine ("DHGS") and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). Additionally, several commercially available preparations of cationic lipids and other components that can be used in the present invention are available. These include, for example, LIPOFECTIN® (a commercially available cationic lipid nanoparticle comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (“DOPE”), GIBCO BRL, Grand Island, NY, USA) and LIPOFECTAMINE® (a commercially available cationic lipid nanoparticle comprising N-(1-(2,3 dioleyloxy)propyl)N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA”) and (“DOPE”), GIBCO BRL).The following lipids are cationic and have a positive charge at subphysiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, DLinKDMA (WO 2009 / 132131), DLin-K-C2-DMA (WO 2010 / 042877), DLin-M-C3-DMA (WO 2010 / 146740 and / or WO 2010 / 105209), DLin-MC3-DMA (heptatriaconta-6,9,28,31tetraen-19-yl 4-(dimethylamino)butanoate; Jayaraman et al. al., 2012, Angew. Chem. Int. Ed. Engl. 51:8529-8533), 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienyloxyl]propan-1-amine) (CLinDMA). Other cationic lipids suitable for use in the present invention include, for example, cationic lipids described in U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, and 5,283,185, and U.S. Patent Application Publication Nos. 2008 / 0085870 and 2008 / 0057080. Other cationic lipids suitable for use in the present invention include, for example, lipids E0001-E0118 or E0119-E0180 disclosed in Table 6 (pages 112-139) of WO 2011 / 076807 (which also discloses methods of making and using these cationic lipids).

[0130] In certain aspects of this embodiment of the invention, the LNPs comprise one or more of the following ionizable cationic lipids: DLinDMA, DlinKC2DMA DLin-MC3-DMA, CLinDMA, or S-Octyl CLinDMA. See International Patent Application Publication No. WO 2010 / 021865.

[0131] In certain aspects of this embodiment of the invention, the LNP comprises one or more ionizable cationic lipids described in International Patent Application Publication No. 2011 / 022460, or any pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds or salts therein.

[0132] Structures of the same constitution that differ in the spatial arrangement of certain atoms or groups are stereoisomers, and topology is an important consideration in analyzing their interrelationships. If the relationship between two stereoisomers is that of an object and its non-superimposable mirror image, the two structures are mirror images, and each structure is said to be chiral. Stereoisomers also include diastereomers, cis-trans isomers, and conformational isomers. Diastereoisomers can be chiral or achiral and are not mirror images of each other. Cis-trans isomers differ only in the position of atoms relative to a specified plane, where these atoms are part of a rigid structure or can be considered as if they were part of a rigid structure. Conformer isomers are formally isomers that can be interconverted by rotation around a single bond. Examples of such conformers include cyclohexane conformations with chair and boat conformers, carbohydrates, and linear alkane conformations with twisted, staggered, and gauche conformers. See J. Org. Chem. 35, 2849 (1970).

[0133] Many organic compounds exist in optically active forms, possessing the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by the compound, with (-) or (-) meaning the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, enantiomers are identical except that they are non-superimposable mirror images of each other. A mixture of enantiomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture. Many of the compounds described herein may have one or more chiral centers and therefore exist in different enantiomeric forms. If desired, a chiral carbon can be indicated with an asterisk (*). When a bond to a chiral carbon is shown as a straight line in the formulae of the present invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and therefore both enantiomers and mixtures thereof, are encompassed within the formula. As used in the art, when it is desired to specify the absolute configuration around a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (a bond to an atom above the plane) and the other as a series of short parallel lines or a wedge (a bond to an atom below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0134] When the compound of the present invention contains one chiral center, the compound exists in two enantiomeric forms, and the present invention includes both enantiomers and mixtures of enantiomers, such as a particular 50:50 mixture called a racemic mixture. Enantiomers can be separated, for example, by crystallization (see CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation (CRC Press, 2001) by David Kozma); by crystallization, by the formation of diastereomeric derivatives or complexes, for example, by gas-liquid or liquid chromatography; by selective reaction of one enantiomer with an enantiomer-specific reagent, for example, enzymatic esterification; or by gas-liquid or liquid chromatography in a chiral environment, for example, on a chiral support, for example, silica with a chiral ligand attached, or in the presence of a chiral solvent. It will be understood that if the desired enantiomer is converted into another chemical entity by one of the above separation procedures, additional steps will be required to isolate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

[0135] The designation of a particular absolute configuration at a chiral carbon of a compound of the present invention is understood to mean that the designated enantiomeric form of the compound is in enantiomeric excess (ee), or in other words, substantially free of other enantiomers. For example, the "R" form of a compound is substantially free from the "S" form of the compound and is thus in enantiomeric excess of the "S" form. Conversely, the "S" form of a compound is substantially free from the "R" form of the compound and is thus in enantiomeric excess of the "R" form. Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%. In certain embodiments, when a particular absolute configuration is designated, the enantiomeric excess of the depicted compound is at least about 90%.

[0136] When a compound of the present invention has two or more chiral carbons, the compound of the present invention can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to four optical isomers and two pairs of enantiomers ((S,S) / (R,R) and (R,S) / (S,R)). Pairs of enantiomers (e.g., (S,S) / (R,R)) are mirror image stereoisomers of each other. Stereoisomers that are not mirror images (e.g., (S,S) and (R,S)) are diastereomers. Pairs of diastereoisomers can be separated by methods known to those skilled in the art, such as chromatography or crystallization, and individual enantiomers within each pair can be separated as described above. The present invention includes each diastereoisomer of such compounds and mixtures thereof.

[0137] LNPs can also contain any combination of two or more of the cationic lipids described herein. In certain embodiments, cationic lipids typically comprise about 0.1 to about 99.9 mol% of the total lipid present in the particle. In certain embodiments, cationic lipids can comprise about 80 to about 99.9% mol%. In other embodiments, cationic lipids comprise about 2% to about 70%, about 5% to about 50%, about 10% to about 45%, about 20% to about 99.8%, about 30% to about 70%, about 34% to about 59%, about 20% to about 40%, or about 30% to about 40% (mol%) of the total lipid present in the particle.

[0138] The LNPs described herein can further include non-cationic lipids, which can be any of a variety of neutral, uncharged, zwitterionic, or anionic lipids capable of forming stable complexes. They are preferably neutral, but can be negatively charged. Examples of non-cationic lipids useful in the present invention include phospholipid-related materials such as natural phospholipids, synthetic phospholipid derivatives, fatty acids, sterols, and combinations thereof. Natural phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacylphosphatidylcholine (lyso-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), N-acyl-PE, phosphoinositides, and phosphosphingolipids. Phospholipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), and phosphatidylserine (DOPS). Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0, and lethicin.

[0139] In certain embodiments of the invention, the non-cationic lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and the like. The non-cationic lipid may be selected from dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). Non-cationic lipids also include sterols such as cholesterol, stigmasterol, or stigmastanol. Cholesterol is known in the art. See U.S. Patent Application Publication Nos. 2006 / 0240554 and 2008 / 0020058. In certain embodiments, the LNP comprises a combination of a phospholipid and a sterol.

[0140] When present, non-cationic lipids typically comprise about 0.1% to about 65%, about 2% to about 65%, about 10% to about 65%, or about 25% to about 65%, expressed as a molar percentage of the total lipids present in the LNP. The LNPs described herein further comprise polyethylene glycol (PEG) lipid conjugates ("PEG-lipids"), which can serve as bilayer-stabilizing components. The lipid component of the PEG-lipid can be any of the non-cationic lipids described above, including natural phospholipids, synthetic phospholipid derivatives, fatty acids, sterols, and combinations thereof. In certain embodiments of the present invention, PEG-lipid comprises, for example, PEG (PEG-DAA) that is bonded to dialkyloxypropyl, as described in International Patent Application Publication No. 05 / 026372, PEG (PEG-DAG) that is bonded to diacylglycerol, as described in US Patent Application Publication Nos. 20030077829 and 2005008689; PEG (PEG-PE) that is bonded to phosphatidylethanolamine (PE), or PEG (PEG-DSG) that is bonded to 1,2-di-O-hexadecyl-sn-glyceride, or any mixture thereof.See, for example, US Patent No. 5,885,613.

[0141] In one embodiment, the PEG-DAG conjugate is a dilaurylglycerol (C12)-PEG conjugate, a PEG-dimyristylglycerol (C14) conjugate, a PEG-dipalmitoylglycerol (C16) conjugate, a PEG-dilaurylglycamide (C12) conjugate, a PEG-dimyristylglycamide (C14) conjugate, a PEG-dipalmitoylglycamide (C16) conjugate, or a PEG-disterylglycamide (C18). Those skilled in the art will readily understand that other diacylglycerols can be used in the PEG-DAG conjugate.

[0142] In certain embodiments, PEG-lipids include, but are not limited to, PEG-dimyristylglycerol (PEG-DMG), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA).

[0143] In certain embodiments, the PEG-lipid is PEG conjugated to dimyristoylglycerol (PEG-DMG), as described, for example, in Abrams et al., 2010, Molecular Therapy 18(1):171, and in U.S. Patent Application Publication Nos. 2006 / 0240554 and 2008 / 0020058.

[0144] In certain embodiments, the PEG-lipid, such as PEG-DAG, PEG-cholesterol, or PEG-DMB, comprises polyethylene glycol having an average molecular weight ranging from about 500 daltons to about 10,000 daltons, about 750 daltons to about 5,000 daltons, about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, or about 2,000 daltons. In certain embodiments, the PEG-lipid comprises PEG400, PEG1500, PEG2000, or PEG5000.

[0145] The acyl group in any of the above lipids is preferably an acyl group derived from a fatty acid having a carbon chain of from about C10 to about C24, hi one embodiment, the acyl group is lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0146] The PEG-lipid conjugate typically comprises about 0.1% to about 15%, about 0.5% to about 20%, about 1.5% to about 18%, about 4% to about 15%, about 5% to about 12%, about 1% to about 4%, or about 2%, expressed as a mole % of the total lipid present in the particle.

[0147] In certain embodiments of the invention, the LNPs comprise one or more cationic lipids, cholesterol, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG).

[0148] In certain embodiments of the invention, the LNPs comprise one or more cationic lipids, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG).

[0149] In some embodiments of the invention, the LNP comprises 34-59 mol% cationic lipid, 30-48 mol% sterol, 10-24 mol% phospholipid, and 1-2 mol% PEG-lipid.

[0150] In some embodiments, the cationic lipid is [Table 4]

[0151] In some embodiments, the sterol is [ka] is.

[0152] In some embodiments, the phospholipid is [Table 5]

[0153] In some embodiments, the polyethylene glycol-lipid is [Table 6]

[0154] In certain embodiments of the invention, the LNPs comprise lipid compounds assembled within the following molar ratios: Cationic lipids (20-99.8 mol%) non-cationic lipids (0.1-65 mol%) and PEG-DMG (0.1–20 mol %).

[0155] In certain embodiments of the invention, the LNPs comprise lipid compounds assembled within the following molar ratios: Cationic lipids (30-70 mol%) non-cationic lipids (20-65 mol%) and PEG-DMG (1–15 mol%).

[0156] In certain aspects of this embodiment, the non-cationic lipid is cholesterol. Exemplary LNPs can comprise cationic lipid / cholesterol / PEG-DMG in approximately the following molar ratio: 58 / 30 / 10.

[0157] In certain aspects of this embodiment, the non-cationic lipid is cholesterol and DSPC. Exemplary LNPs can comprise cationic lipid / cholesterol / DSPC / PEG-DMG in approximately the following molar ratios: 59 / 30 / 10 / 1; 58 / 30 / 10 / 2; 43 / 41 / 15 / 1; 42 / 41 / 15 / 2; 40 / 48 / 10 / 2; 39 / 41 / 19 / 1; 38 / 41 / 19 / 2; 34 / 41 / 24 / 1; and 33 / 41 / 24 / 2.

[0158] Preparation of LNPs LNPs can be formed by a rapid precipitation process involving micromixing of lipid components dissolved in ethanol with an aqueous solution using a confined-volume mixing device, such as a confined-volume T-mixer, a multi-inlet vortex mixer (MIVM), or a microfluidic mixer device, as described below. The lipid solution contains one or more cationic lipids, one or more non-cationic lipids (e.g., DSPC), PEG-DMG, and optionally cholesterol, in ethanol at specific molar ratios. The aqueous solution consists of a sodium citrate or sodium acetate buffered salt solution with a pH ranging from 2 to 6, preferably 3.5 to 5.5. The two solutions are heated to a temperature ranging from 25°C to 45°C, preferably 30°C to 40°C, and then mixed in the confined-volume mixer, thereby immediately forming LNPs. When a confined-volume T-mixer is used, the T-mixer has an inner diameter (ID) ranging from 0.25 to 1.0 mm. The alcohol and aqueous solutions are delivered to the inlet of a T-mixer using a programmable syringe pump at a total flow rate of 10-600 mL / min. The alcohol and aqueous solutions are combined in a very small volume mixer at a ratio within the range of 1:1 to 1:3 volume:volume, with a target ratio of 1:1.1 to 1:2.3. The combination of ethanol volume fraction, reagent solution flow rate, and T-mixer tubing ID utilized in this mixing step has the effect of adjusting the LNP particle size between 30 and 300 nm. In a sequential, multi-stage in-line mixing process, the resulting LNP suspension is diluted twice into a buffer solution with a higher pH ranging from 6 to 8. For the first dilution, the LNP suspension is mixed with a buffer solution with a higher pH (pH 6-7.5) at a mixing ratio within the range of 1:1 to 1:3 volume:volume, with a target ratio of 1:2 volume:volume. This buffer solution is maintained at a temperature within the range of 15-40 °C, with a target temperature of 30-40 °C. The resulting LNP suspension is further mixed with a buffer solution at a higher pH, e.g., 6-8, and at a volume:volume ratio ranging from 1:1 to 1:3, with a target of 1:2. The temperature of the buffer solution is then between 15-40°C, with a target of 16-25°C. The mixed LNP is allowed to stand for 30 minutes to 2 hours before the anion exchange filtration step.The temperature during the incubation period ranges from 15 to 40°C, with a target of 30 to 40°C. After incubation, the LNP suspension is filtered through a 0.8 μm filter that includes an anion exchange separation step. This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and flow rates of 10 to 2000 mL / min. The LNP are concentrated and diafiltered via an ultrafiltration process to remove alcohol and exchange the buffer for a final buffer, such as phosphate-buffered saline or a buffer system suitable for cryopreservation (e.g., containing sucrose, trehalose, or a combination thereof). The ultrafiltration process uses a tangential flow filtration format (TFF). This process uses a membrane nominal molecular weight cutoff range of 30 to 500 KD, with a target of 100 KD. The membrane format can be hollow fiber or flat-sheet cassette. A TFF process with the appropriate molecular weight cutoff retains the LNP in the retentate, while the filtrate or permeate contains the alcohol and final buffer waste. The TFF process is a multistep process involving initial concentration to a lipid concentration of 20–30 mg / mL. After concentration, the LNP suspension is diafiltered against a final buffer (e.g., phosphate-buffered saline (PBS) pH 7–8, 10 mM Tris, 140 mM NaCl pH 7–8, or 10 mM Tris, 70 mM NaCl, 5 wt% sucrose, pH 7–8) in 5–20 volumes to remove alcohol and perform buffer exchange. The material is then further concentrated 1–3x via ultrafiltration. The final step in the LNP manufacturing process is sterile filtration of the concentrated LNP solution into an appropriate container under aseptic conditions. Sterile filtration is achieved by passing the LNP solution through a prefilter (Acropak 500 PES 0.45 / 0.8 μm capsule) and a bioburden-reducing filter (Acropak 500 PES 0.2 / 0.8 μm capsule). After filtration, store the vialed LNP product under appropriate storage conditions (2 °C to 8 °C, or -20 °C for frozen formulations).

[0159] In some embodiments, the LNPs of the compositions provided herein have a mean geometric diameter of less than 1000 nm. In some embodiments, the LNPs have a mean geometric diameter of more than 50 nm but less than 500 nm. In some embodiments, the mean geometric diameter of a population of LNPs is about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm. In some embodiments, the mean geometric diameter is 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometric diameter is 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the mean geometric diameter is 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs in a population of LNPs have a diameter less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs in a population of LNPs have a diameter greater than 50 nm but less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs in a population of LNPs have a diameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs in a population of LNPs have a diameter of 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm.In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs in a population of LNPs have a diameter of 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm.

[0160] In certain embodiments, the size of the LNPs ranges from about 1 to 1000 nm, preferably about 10 to 500 nm, more preferably about 100 to 300 nm, preferably 100 nm.

[0161] Therapeutic and Prophylactic Compositions Provided herein are vaccine compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, and / or diagnosis of norovirus in humans and other mammals. Norovirus vaccines can be used as therapeutic or prophylactic drugs. Norovirus vaccines can be used in medicine to prevent and / or treat infectious diseases. In some embodiments, vaccines according to the present disclosure can be used for the treatment of norovirus.

[0162] Provided herein are pharmaceutical compositions comprising a Norovirus vaccine, optionally in combination with one or more pharmaceutically acceptable excipients.

[0163] Norovirus vaccines can be formulated or administered alone or in combination with one or more other components. For example, norovirus vaccines (vaccine compositions) can include other components, including, but not limited to, adjuvants.

[0164] In some embodiments of the invention provided herein, the norovirus vaccine does not contain an adjuvant (i.e., is adjuvant-free). In other embodiments, the norovirus vaccine comprises lipid- or polymer-based nanoparticles. In certain embodiments, any of the lipid nanoparticles described herein can act as an adjuvant when combined with a norovirus vaccine.

[0165] Aluminum has previously been shown to stimulate immune responses to co-administered antigens primarily by stimulating TH2 responses. Preferably, the aluminum adjuvant in the compositions provided herein is not in the form of an aluminum precipitate. Aluminum-precipitated vaccines can increase immune responses to target antigens, but they have been shown to be highly heterogeneous preparations and have had inconsistent results (see Lindblad EB Immunology and Cell Biology 82:497-505 (2004)). In contrast, aluminum-adsorbed vaccines can be pre-prepared in a standardized manner, which is an essential feature of vaccine preparations for human administration. Furthermore, physical adsorption of the desired antigen onto the aluminum adjuvant is thought to play an important role in adjuvant function, possibly in part by allowing for slower clearance from the injection site or more efficient uptake of the antigen by antigen-presenting cells.

[0166] The aluminum adjuvants of the present invention can be in the form of aluminum hydroxide (Al(OH)), aluminum phosphate (AlPO), aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS), or so-called "alum" (KAl(SO)-12H0). See Klein et al., "Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27) A1 MAS NMR." J. Pharm. Sci. 89(3):311-21 (2000).

[0167] In some embodiments of the invention provided herein, the aluminum adjuvant is aluminum hydroxyphosphate or AAHS. The phosphate to aluminum ratio in the aluminum adjuvant can range from 0 to 1.3. In some embodiments of this aspect of the invention, the phosphate to aluminum ratio is within the range of 0.1 to 0.70. In certain embodiments, the phosphate to aluminum ratio is within the range of 0.2 to 0.50. APA is an aqueous suspension of aluminum hydroxyphosphate. APA is produced by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the mixing process, the material is sized in a high-shear mixer to achieve a target aggregate particle size in the range of 2 to 8 μm. The product is then diafiltered against saline and steam sterilized. See, e.g., International Patent Application Publication No. WO 2013 / 078102.

[0168] In some embodiments of the invention, the aluminum adjuvant is in the form of AAHS (interchangeably referred to herein as Merck Aluminum Adjuvant (MAA)). MAA has a zero charge at neutral pH, whereas AlOH has a net positive charge and AlPO typically has a net negative charge at neutral pH.

[0169] Those skilled in the art will be able to determine the optimal dosage of aluminum adjuvant that is safe and effective in increasing the immune response to the targeted antigenic polypeptide. For a discussion of aluminum's safety profile and the amount of aluminum contained in FDA-approved vaccines, see Baylor et al., Vaccine 20:S18-S23 (2002). Generally, effective and safe doses of aluminum adjuvant range from 150 to 600 μg per dose (300 to 1200 μg / mL concentration). In certain embodiments of the formulations and compositions of the present invention, 200 to 300 μg of aluminum adjuvant is present per vaccine dose. In other embodiments of the formulations and compositions of the present invention, 300 to 500 μg of aluminum adjuvant is present per vaccine dose.

[0170] Norovirus vaccine formulations and methods of use The norovirus vaccines of the present disclosure can 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 active agent, a prophylactically active agent, or a combination of both. The vaccine composition can 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.

[0171] Formulations of the norovirus vaccine compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparative methods include bringing into association an active ingredient (e.g., one or more polynucleotides) with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desired, dividing, shaping, and / or packaging the product into desired single or multiple dose units.

[0172] 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 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.

[0173] Some embodiments of the present disclosure provide formulations of norovirus vaccines, which are formulated in an amount effective to produce an antigen-specific immune response in a subject (e.g., the production of antibodies specific to norovirus antigenic polypeptides). The "effective amount" of a norovirus RNA vaccine is provided based, at least in part, on the target tissue, target cell type, administration method, physical characteristics of the norovirus RNA vaccine's polynucleotide (e.g., size and degree of modified nucleosides) and other components, and other determinants. Generally, an effective amount of a norovirus RNA vaccine composition will induce or boost an immune response, preferably in response to intracellular antigen production, more efficiently than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production may 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., as demonstrated by an increased duration of protein translation from a modified polynucleotide), or an altered antigen-specific immune response of the host cell.

[0174] In some embodiments, the antigen-specific immune response is characterized by measuring the anti-norovirus antigenic polypeptide antibody titer generated in a subject administered a norovirus vaccine provided herein. The antibody titer is a measure of the amount of antibodies in a subject, e.g., antibodies specific to a particular antigen (e.g., a norovirus antigenic polypeptide such as a VP1 or VP2 polypeptide) or epitope of an antigen. The antibody titer is typically expressed as the reciprocal of the highest dilution that produces a positive result. Enzyme-linked immunosorbent assay (ELISA), for example, is a common assay for determining antibody titer.

[0175] In some embodiments, antibody titers are used to assess whether a subject has had an infectious disease or to determine whether immunization is required. In some embodiments, antibody titers are used to determine the strength of an autoimmune response, whether a booster immunization is required, whether a previous vaccine was effective, and to identify any recent or previous infections. According to the present disclosure, antibody titers can be used to determine the strength of the immune response induced in a subject by a norovirus vaccine.

[0176] Mode of vaccine administration Norovirus vaccines can be administered prophylactically or therapeutically as part of an active immunization scheme for healthy individuals, or can be administered early in infection during the incubation period or during active infection after the onset of symptoms. A prophylactically effective dose is a therapeutically effective dose that prevents infection by the virus at a clinically acceptable level. In some embodiments, a therapeutically effective dose is the dose listed in the vaccine package insert. In some embodiments, the amount of the vaccine of the present disclosure administered to a cell, tissue, or subject can be an amount effective for immunoprophylaxis.

[0177] The norovirus vaccine can be formulated for administration by any route that produces a therapeutically effective outcome, including, but not limited to, intradermal, intramuscular, intranasal, intratracheal, and / or subcutaneous administration. In some embodiments, the norovirus vaccine is administered intramuscularly.

[0178] The present disclosure provides methods comprising administering a vaccine to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc.

[0179] Norovirus vaccine compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage.However, it is understood that the total daily use amount of vaccine compositions can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective or suitable diagnostic imaging dose level for any specific patient / animal will depend on various factors, including the disorder to be treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and diet of the patient / animal; the administration time, administration route and excretion rate of the specific compound used; the duration of treatment; the drugs used in combination with or simultaneously with the specific compound used; and similar factors well known in the medical field.

[0180] The norovirus vaccine may be administered 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" refers to an additional administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The interval between the first administration of the prophylactic composition and the booster may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 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 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, The period of time between the first administration of the prophylactic composition and the booster can be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 7 years, 8 months, 9 years, 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 more than 99 years. In some embodiments, the time between the first administration of the prophylactic composition and the booster can be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0181] Norovirus vaccines can be used in a variety of settings, depending on the prevalence of infectious diseases or the degree or level of medical need for diseases for which no treatment has been established. As a non-limiting example, norovirus vaccines can be used to treat and / or prevent various noroviruses. Vaccines have superior properties in that they produce much greater antibody titers and generate a response more quickly than commercially available antiviral agents and compositions.

[0182] Treatment method Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of norovirus in humans and other mammals. Norovirus vaccines can be used as therapeutic or prophylactic agents. Norovirus vaccines can be used in medicine to prevent and / or treat infectious diseases. In exemplary embodiments, the norovirus vaccine of the present disclosure is used to provide prophylactic protection from norovirus. Prophylactic protection from norovirus can be achieved after administration of the norovirus vaccine of the present disclosure. The vaccine can be administered once, twice, three times, four times, or more times. Although less desirable, it is possible to administer the vaccine to infected individuals to achieve a therapeutic response. Dosage may need to be adjusted accordingly.

[0183] In some embodiments, the norovirus vaccines of the present disclosure can be used as methods for preventing a norovirus infection in a subject, comprising administering at least one norovirus vaccine provided herein to the subject. In some embodiments, the norovirus vaccines of the present disclosure can be used as methods for inhibiting a primary norovirus infection in a subject, comprising administering at least one norovirus vaccine provided herein to the subject. In some embodiments, the norovirus vaccines of the present disclosure can be used as methods for treating a norovirus infection in a subject, comprising administering at least one norovirus vaccine provided herein to the subject. In some embodiments, the norovirus vaccines of the present disclosure can be used as methods for reducing the incidence of norovirus infection in a subject, comprising administering at least one norovirus vaccine provided herein to the subject. In some embodiments, the norovirus vaccines of the present disclosure can be used as a method for inhibiting the transmission of norovirus from a first subject infected with norovirus to a second subject not infected with norovirus, the method comprising administering at least one norovirus vaccine provided herein to at least one of the first and second subjects.

[0184] A method of eliciting an immune response against Norovirus in a subject is provided in accordance with an embodiment of the present invention, comprising administering to the subject a Norovirus vaccine described herein, thereby inducing in the subject an immune response specific to a Norovirus antigenic polypeptide or immunogenic fragment thereof.

[0185] [Example] The following examples are intended to be illustrative and should not be construed as further limiting. All references, patents and published patent applications cited throughout this application, including the figures, are hereby expressly incorporated by reference.

[0186] [Example 1] Preparation of mRNA DNA sequences encoding the norovirus antigenic VP1 protein were prepared and used for subsequent RNA in vitro transcription reactions. VP1 protein sequences were obtained from public databases (GenBank, INSDC), and a single consensus sequence was selected for each genotype based on the most common recent sequences (i.e., GenBank ID QBW96040 for GII.4, GenBank ID NP_056821 for GI.1, GenBank ID AWT08315 for GII.6, GenBank ID YP_009518839 for GII.2, and GenBank ID QJF54133 for GII.3). The selected DNA sequences were modified by introducing codon-modified or GC-optimized sequences for stabilization. The DNA sequences were altered from the native sequence as follows: Silent mutations were introduced to maximize the codon adaptation index (CAI). See Sharp PM, Li WH. The Codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987 Feb 11;15(3):1281-95. The second most frequent codon was used to disrupt runs of five or more identical nucleotides. Furthermore, DNA 30-mers with a G / C content greater than 80% were mutated to reduce the G / C content to 80% or less using the second most frequent codons: Pro (CCC->CCA), Gly (GGC->GGA), and / or Ala (GCC->GCA). For example, using a short amino acid sequence "proline-proline," such as that found in GII.3 DNA, (1) the native sequence CCC-CCT was first mutated to CCC-CCC to optimize CAI, and then (2) further modified to the second most frequent codon for proline to avoid homopolymer runs. The codon-optimized DNA sequences are shown as SEQ ID NOs: 9-13 in Table 1 below.

[0187] [Table 7] TIFF2026505939000011.tif233168 TIFF2026505939000012.tif234166 TIFF2026505939000013.tif233167 TIFF2026505939000014.tif235168 TIFF2026505939000015.tif234166 TIFF2026505939000016.tif232167 TIFF2026505939000017.tif233167 TIFF2026505939000018.tif233164 TIFF2026505939000019.tif232168 TIFF2026505939000020.tif233167 TIFF2026505939000021.tif234166 TIFF2026505939000022.tif32166

[0188] [Example 2] Preparation of purified mRNA of five norovirus VP1-encoding sequences Five purified DNA plasmids were produced by GenScript Probio Biotech and constructed in a pUC57 backbone containing a T7 promoter sequence, the encoded 5' UTR sequence (SEQ ID NO: 6), the GII.4, GI.1, GII.6, GII.2, or GII.3 norovirus VP1 DNA sequence (SEQ ID NOs: 9-13) encoding the GII.4, GI.1, GII.6, GII.2, or GII.3 norovirus VP1 mRNA CDS (SEQ ID NOs: 1-5), and the encoded 3' UTR sequence (SEQ ID NO: 7) with a poly(A) tail. The DNA plasmids were linearized using BbsI-HF restriction enzyme (New England Biolabs) for the GII.4, GII.6, GII.2, or GII.3-encoding plasmids (SEQ ID NOs: 9, 11-13) and BspQI restriction enzyme (New England Biolabs) for the GI.1-encoding plasmid (SEQ ID NO: 10). Approximately 1 mg of plasmid was added to the linearization reaction mixture at a concentration of approximately 0.2–0.4 mg / mL along with 1–2 U / mL of the appropriate restriction enzyme (New England Biolabs) in digestion buffer and incubated at 37°C for approximately 1 hour. The linearization reaction was purified by isopropanol and ethanol precipitation steps to produce purified linear DNA template at a concentration of approximately 0.8–1.2 mg / mL. Five IVT reactions were performed at a 10 mL scale in 60 mL high-density polyethylene (HDPE) bottles (Nalgene) mixed in a shaking incubator (Benchmark Scientific) set at a temperature of approximately 37°C. The purified linear DNA template was added to the IVT reaction mixture at a concentration of approximately 40–60 μg / mL. ATP, GTP, and CTP (New England Biolabs) were added to the IVT reaction mixture at concentrations ranging from approximately 8–14 mM, and UTP was added to the IVT reaction mixture at a concentration of approximately 100 mM. 1 Completely replaced by Ψ(BOC Sciences), m 1Ψ (BOC Sciences) was added at approximately 4-6 mM, and CleanCap AG(3'OMe) (TriLink Biotechnologies) was added at approximately 3-5 mM. T7 RNA polymerase (New England Biolabs) was added to the IVT reaction mixture at a concentration of approximately 10,000-12,000 U / mL, yeast inorganic pyrophosphatase (New England Biolabs) was added at approximately 2 U / mL, and mouse RNase inhibitor (New England Biolabs) was added at 1 U / mL. The IVT reaction mixture was prepared in a transcription buffer matrix similar to 1x T7 Transcription Buffer (New England Biolabs), containing Tris-HCl, magnesium, spermidine, DTT, and other excipients at a final solution pH of approximately 6-9. The IVT reaction mixture was incubated for 3–4 h with mixing, and the reaction was stopped by adding DNase I (New England Biolabs) at a concentration range of approximately 350 U / mL along with calcium chloride (Sigma Aldrich) at a concentration of 3.5 mM and incubated at 37°C for 1 h.

[0189] The five IVT reaction mixtures were quenched with approximately 50 mM EDTA (Thermo Fisher Scientific) and diluted to approximately 300 mL in an oligo-dT binding matrix consisting of 400 mM sodium chloride (Thermo Fisher Scientific), 10 mM Tris-HCl (Thermo Fisher Scientific), 2 mM EDTA, pH 7.2. The five diluted IVT reaction mixtures were purified using a 40 mL CIMmultus oligo-dT monolithic column (Sartorius AG) with a residence time of approximately 0.5 minutes. After hybridization of the loaded mRNA with a poly(A) tail to the oligo-dT ligand, the column was washed with 5 CV of a buffer consisting of 50 mM sodium chloride, 10 mM Tris-HCl, 2 mM EDTA, pH 7.2, and the bound mRNA was eluted with 5 CV of 10 mM Tris-HCl, pH 7.2. The five oligo-dT elution fractions were collected at 235 cm. 2Load the membrane onto a 50 kDa hollow fiber PES membrane (Repligen) and incubate at approximately 4 psi for 4,000 s. -1 The 6DV was concentrated approximately 8-fold and buffer exchanged into 1 mM sodium citrate (Thermo Fisher Scientific), pH 6.4, operating at a cross-flow shear rate of 18 cm. 2 300 L / m through a 0.2 μm PVDF (Millipore) bioburden reduction filter. 2 The final TFF retentate was pumped at a flux of 0.1-hr. Bioburden-reduced filter product mRNA concentrations were calculated from sample absorbance measurements at 260 nm using a spectrophotometer. The product was diluted to approximately 1 mg / mL with 1 mM sodium citrate, pH 6.4, filled into conical tubes, and stored at <-60°C. The final mRNA yields purified from 10 mL IVT reactions were 85 mg, 78 mg, 89 mg, 75 mg, and 78 mg for GII.4, GI.I, GII.6, GII.2, and GII.3 mRNAs, respectively. Purified mRNAs were analyzed by agarose gel electrophoresis using a 2% agarose E-Gel and Power Snap electrophoresis system (Thermo Fisher Scientific), and the expected size and purity were observed. Figure 1 shows an image of a 2% agarose E-gel of purified mRNA for five norovirus VP1 coding sequences at a loading of 50 ng of mRNA per lane. Lanes 1-2 and 10-12 are blanks, lanes 3 and 9 contain ssRNA ladder (New England Biolabs), lane 4 contains GII.I, lane 5 contains GII.2, lane 6 contains GII.3, lane 7 contains GII.4, and lane 8 contains GII.6.

[0190] [Example 3] LNP encapsulation of mRNA Preparation of mRNA / LNPs LNPs encapsulating one or up to five different norovirus mRNA constructs (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) can be formed by rapid precipitation using a microfluidics mixer, multi-inlet vortex mixer, or T-mixer to micromix two fluid streams. One fluid stream contains lipids dissolved in ethanol, and the other is an aqueous solution containing mRNA. The lipid solution prepared in ethanol contains cationic lipid, cholesterol, PEG-DMG, and phospholipid (DSPC) in specific molar ratios and is heated to a temperature ranging from 25 to 45 °C. The mRNA solution, prepared to achieve a target mRNA weight percentage, consists of a sodium citrate buffered salt solution with a pH ranging from 4 to 6 and is maintained at a temperature ranging from 16 to 25 °C. The lipid molar ratio and mRNA weight percentage are selected to achieve a nitrogen-to-phosphate (N / P) ratio ranging from 2 to 9, with a target of 5 to 7. The lipid and mRNA solutions are micromixed to instantly form LNPs encapsulating mRNA (mRNA / LNPs).

[0191] For microfluidic mixed mRNA / LNP, combine the mRNA and lipid-containing solutions at a ratio of 1:1 to 5:1 at 8-15 mL / min to produce 16-50% volume:volume alcohol in the mixed solution and achieve an N / P ratio ranging from 2 to 9, with a target of 5-7. The resulting mRNA / LNP suspension then undergoes buffer exchange and alcohol removal by dialysis. The mRNA / LNP suspension is dialyzed 2-4 times against a final buffer solution in a pH range of 7-8. The final buffer solution may contain a cryoprotectant (e.g., containing sucrose, trehalose, or a combination). The dialysis step can use a membrane nominal molecular weight cutoff range of 30-500 kDa. Prior to final sterile filtration, the mRNA / LNP suspension may be combined with additional norovirus mRNA / LNP suspensions encoding VP1 proteins from different norovirus genotypes (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) to form co-formulated bivalent, trivalent, tetravalent, or pentavalent products. The monovalent or co-formulated mRNA / LNP suspensions are then filtered through 0.2 μm polyethersulfone (PES) or polyvinylidene fluoride (PVDF) sterile filters into sterile glass vials and sealed. The resulting mRNA / LNP formulations can then be stored under refrigerated (2-8 °C) or frozen (-20 ± 10 °C or ≤ -60 °C) conditions. When monovalent mRNA / LNP formulations are prepared in sterile vials, these formulations can be equilibrated to temperatures ranging from 16-25°C and, prior to administration, combined with additional norovirus mRNA / LNP formulations encoding VP1 proteins from different norovirus genotypes (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) to form co-formulated bivalent, trivalent, tetravalent, or pentavalent final vaccines.

[0192] For micromixing using a very small-volume T-mixer, the T-mixer has an inner diameter (ID) of 0.25-1.0 mm. The lipid and mRNA solutions are delivered to the inlet of the T-mixer using a programmable pump. The total flow rate at the inlet of the T-mixer ranges from 100-600 mL / min. The lipid and mRNA solutions are delivered to the T-mixer at ratios ranging from 0.9:1 to 1:3 volume:volume, with a target range of 1:1.1 to 1:2, to produce 16-53% volume:volume alcohol in the mixed solution and achieve an N / P ratio ranging from 2-9, with a target range of 5-7. Using a sequential, multi-stage in-line mixing process, the resulting mRNA / LNP suspension is diluted twice into a higher pH buffer solution ranging from 6-8. The mRNA / LNP suspension is first diluted with a higher pH (6-8) buffer solution using a mixing ratio ranging from 1:1 to 1:3 volume:volume, with a target of 1:2 volume:volume. This buffer solution is at a temperature within the range of 15-40°C, with a target temperature of 30-40°C. The second dilution involves mixing the mRNA / LNP suspension with a buffer solution of a higher pH (pH 6-8) using a mixing ratio ranging from 1:1 to 1:3 volume:volume, with a target of 1:2 volume:volume. The second dilution buffer solution is at a temperature within the range of 15-40°C, with a target temperature of 16-25°C. Before undergoing anion exchange filtration, the fully diluted mRNA / LNP suspension is kept at a temperature within the range of 15-40°C, with a target temperature of 16-25°C, for 30 minutes to 4 hours. After the incubation step is complete, the mRNA / LNP suspension is filtered through a 0.8 μm anion exchange filter using tubing with an ID ranging from 1-10 mm and a flow rate ranging from 10-2000 mL / min. The mRNA / LNP is concentrated and diafiltered using ultrafiltration in a tangential flow filtration (TFF) format to remove alcohol. The mRNA / LNP suspension is then buffer exchanged into a final buffer solution with a pH range of 7-8. The final buffer solution may be suitable for cryopreservation (e.g., containing sucrose, trehalose, or a combination). The TFF process can use hollow fiber or flat sheet membranes with a membrane nominal molecular weight cutoff range of 30-500 kDa, with a target of 500 kDa, to retain the mRNA / LNP in the retentate.The multi-step TFF process begins with a concentration step to achieve an mRNA concentration of 0.4–0.7 mg / mL, followed by diafiltration against a final buffer (e.g., modified Dulbecco's phosphate-buffered saline (mDPBS) with a pH of 7–8 or 10 mM Tris, 10% w / v sucrose, pH 7–8) in 5–20 volumes to remove alcohol and perform buffer exchange. Ultrafiltration is then used to further concentrate the resulting mRNA / LNP suspension 1–5x. Once concentrated, the mRNA / LNP suspension undergoes bioburden-reducing filtration using sequential 0.45 µm and 0.2 µm PES, cellulose acetate (CA), and / or PVDF filters. The bioburden-reduced mRNA / LNP suspension is then stored under refrigerated (2–8 °C) or frozen (≤-60 °C) conditions until further use. Prior to final sterile filtration, the mRNA / LNP suspension may be thawed in a water bath at temperatures ranging from 20 to 30 °C and combined with additional norovirus mRNA / LNP suspensions specifically encoding VP1 proteins from different norovirus genotypes (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) to form co-formulated bivalent, trivalent, tetravalent, or pentavalent products. The resulting mRNA / LNP suspension can then be diluted to the target mRNA concentration and filled into vials before undergoing final sterile filtration using sequential 0.45 μm and 0.2 μm PES and / or PVDF filters. The final sterile-filtered mRNA / LNP suspension can also be lyophilized. The final mRNA / LNP vials (liquid or lyophilized) are then stored under refrigerated (2 to 8 °C) or frozen (-20 ± 10 °C or ≤ -60 °C) conditions. The final sterilized monovalent norovirus mRNA / LNP formulation (GI.1, GII.2, GII.3, GII.4, or GII.6) in a sterile vial can be thawed or reconstituted with a diluent (e.g., saline) and combined with additional norovirus mRNA / LNP formulations encoding VP1 proteins from different norovirus genotypes (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) to form a co-formulated bivalent, trivalent, tetravalent, or pentavalent final vaccine for administration.

[0193] Preparation of mRNA / LNP formulations LNPs encapsulating one norovirus mRNA construct were formed by rapid precipitation using microfluidics or a T-mixer to mix two fluid streams. One fluid stream contained lipids dissolved in ethanol, and the other was an aqueous solution containing mRNA. The lipid solution prepared in ethanol contained cationic lipids, cholesterol, PEG-DMG, and phospholipid (DSPC) in specific molar ratios. The aqueous solution consisted of 10 mM sodium citrate buffer, pH 4.5–5.5, and contained mRNA.

[0194] For microfluidic mixed LNPs, the mRNA and lipid-containing solution flows were combined at a 3:1 ratio at 12 mL / min to produce 25% v:v alcohol in the mixed solution. The resulting LNP suspension had an N / P ratio ranging from 5 to 8. The mRNA / LNP suspension was subjected to buffer exchange and ethanol removal by dialysis. Using SpectraPor® Float-A-Lyzers® (Repligen) with a 100 kDa molecular weight cutoff, the mRNA / LNP suspension was dialyzed three times against 20 mM Tris, 10% sucrose (w / v), pH 7.5, at a volume 100–200 times the product volume. The dialyzed mRNA / LNP suspension was then filtered through a 0.2 μm polyethersulfone (PES; Pall) or polyvinylidene fluoride (PVDF; Millipore) sterile filter into a sterile glass vial and sealed. The mRNA / LNP formulation can then be stored under refrigerated (2-8°C) or frozen (20°C or ≤-60°C) conditions. Prior to use, the mRNA / LNP vial is equilibrated to room temperature and used as a monovalent vaccine or co-formulated with additional monovalent norovirus mRNA / LNP formulations encoding VP1 proteins from different norovirus genotypes (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) to form co-formulated bivalent, trivalent, tetravalent, or pentavalent vaccines.

[0195] For mRNA / LNPs prepared in a t-mixer, the lipid-containing solution stream was heated to a temperature ranging from 35 to 40 °C and then mixed with the mRNA-containing solution stream in a very small volume mixer (ID 0.5 mm) using a programmable pump. The total flow rate at the inlet of the t-mixer ranged from 100 to 150 mL / min, delivering the streams at a ratio of 0.90:1 to 1.3:1, resulting in 43 to 53% vol-vol alcohol in the mixed solution. The resulting mRNA / LNP suspensions, with N / P ratios ranging from 5 to 8, were sequentially diluted two-fold into higher pH buffers (pH range 6 to 8) using a multi-stage in-line mixing process. For the first dilution, the mRNA / LNP formulation was mixed with 20 mM sodium citrate, 300 mM sodium chloride, pH 6, at a 1:1 vol:vol mixing ratio. The buffer solution was maintained at a temperature ranging from 35 to 40 °C. The mRNA / LNP formulation was then further mixed with mDPBS, pH 7.5, at a 1:1 volume:volume ratio. The mDPBS, pH 7.5 solution was at a temperature ranging from 16 to 25°C. The resulting mRNA / LNP suspension was maintained at a temperature ranging from 16 to 25°C for 30 minutes before undergoing anion exchange filtration. After filtration, the mRNA / LNP suspension was then concentrated and diafiltered via an ultrafiltration process to remove alcohol and perform buffer exchange to the final buffer. TFF was used for the ultrafiltration process. A PES membrane using a hollow fiber format with a nominal molecular weight cutoff of 500 kDa was used for ultrafiltration. First, the mRNA / LNP suspension was concentrated 6-8 times by volume using ultrafiltration, targeting an mRNA concentration of 0.4-0.7 mg / mL. The alcohol was removed by subsequent diafiltration (10 diavolumes) using 20 mM Tris, 10% sucrose (w / v), pH 7.5. The mRNA / LNP suspension was then further concentrated 3x by volume, targeting an mRNA concentration of 1.0-1.5 mg / mL. Once concentrated, the monovalent mRNA / LNP formulation underwent bioburden-reducing filtration using sequential 0.45 μm and 0.2 μm PES, CA, and / or PVDF filters. The bioburden-reduced monovalent mRNA / LNP suspension was then stored under frozen (≦-60°C) conditions until further use.Prior to final sterile filtration, the mRNA / LNP suspension was thawed using a water bath at a temperature range of 20-30°C. The thawed mRNA / LNP suspension was then sterile filtered using sequential 0.45 μm and 0.2 μm PES and / or PVDF filters. After filtration and under aseptic conditions, the mRNA / LNP suspension was diluted to the final target mRNA concentration using sterile 20 mM Tris, 10% sucrose (w / v), pH 7.5, filled into sterile vials, and sealed. The sealed mRNA / LNP-containing vials were then stored under refrigerated conditions (≤-60°C). Prior to administration, the mRNA / LNP vials were equilibrated to room temperature and used as monovalent vaccines or co-formulated with additional monovalent norovirus mRNA / LNP formulations encoding VP1 proteins from different norovirus genotypes (e.g., GI.1, GII.2, GII.3, GII.4, and / or GII.6) to form co-formulated bivalent, trivalent, tetravalent, or pentavalent vaccines.

[0196] [Example 4] In vivo immunization with mRNA encapsulated in LNPs Mouse studies This study was designed to test the immunogenicity of candidate norovirus vaccines in mice. The animals tested were 6- to 8-week-old BALB / c mice obtained from Charles River Laboratories. The test vaccine contained mRNA sequences encoding the VP1 proteins of five norovirus genotypes, GI.1, GII.2, GII.3, GII.4, and GII.6, formulated in LNP. Control animals were vaccinated with empty LNP.

[0197] At weeks 0 and 4, animals were immunized intramuscularly with a total volume of 100 μL of each test vaccine, administered in a 50 μL immunization in each quadriceps muscle. The candidate vaccines evaluated in this study are described above and outlined in Table 2 below. Serum was collected from all animals. Route of Administration (ROA).

[0198] [Table 8]

[0199] To test for the presence of antibodies capable of binding to norovirus-derived VLPs, 384-well ELISA plates were coated with 50 ng / well of one of the following VLPs corresponding to norovirus genotypes: GI.1, GII.2, GII.3, GII.4, and GII.6. During coating, a liquid handler prepared 10-point, 4-fold serial dilutions of serum in blocking buffer. After coating, the ELISA plate was washed, incubated with blocking buffer, emptied, and then incubated with samples from the serum dilution plate. After a 2-hour incubation, the ELISA plate was washed, and goat anti-mouse IgG(Fc)-HRP conjugate was added to each well. The plate was incubated for 1 hour, washed, and incubated with chemiluminescent substrate for 15 minutes before luminescence readings (ultrasensitive, 0.1 seconds per well) were taken on a plate reader. The interpolated endpoint titer was calculated as the highest dilution at which the relative luminescence (RLU) signal exceeded the 50,000 RLU cutoff. Samples whose dilutions did not exceed the threshold were given a titer of "25," and for samples that exceeded the threshold at the highest dilution tested—1:13,107,200—a value of 13,107,200 was used for the titer. Samples whose well value was exactly 50,000 RLU were given the interpolated titer value at that exact dilution. Serum antibody titers were plotted as geometric mean titers with 95% confidence intervals using GraphPad Prism software.

[0200] Figures 2A-2E illustrate interpolated endpoint titers of serum samples from animals vaccinated with the test vaccines. The vaccine groups tested are indicated on the x-axis, and binding to VLPs from each of the different norovirus genotypes is plotted. The dotted line at 50 indicates the limit of detection. A value of 25 indicates no binding, even at the lowest dilution tested. Compared to the monovalent vaccine candidates, administration of the pentavalent vaccine induced comparable serum IgG titers specific for the five norovirus genotypes GI.1, GII.2, GII.3, GII.4, and GII.6. A second dose of all monovalent and pentavalent vaccines boosted antibody titers by week 6.

[0201] To explore functional antibody responses, the ability of serum from week 6 (after dose 2) to block VLP binding to HBGA was assessed. Depending on the test genotype, 384-well HBGA plates were coated with 250 ng / well of porcine gastric mucin (PGM) or 1:1000 diluted human saliva. During coating, a liquid handler prepared 10-point, 2-fold serial dilutions of serum in blocking buffer. After dilution, 30 μL / well of GI.1, GII.2, GII.3, GII.4, or GII.6 VLPs was added to each sample. After coating, the HBGA plates were washed, incubated with blocking buffer, emptied, and incubated with the mixture from the serum dilution + VLP plate. After a 2-hour incubation, the plates were washed, and rabbit serum hyperimmunized with the corresponding VLP was added to each well. After a 1-hour incubation, the plates were washed, and goat anti-rabbit IgG (Fc fragment-specific)-HRP was added. Plates were incubated for 1 hour, washed, and incubated with chemiluminescent substrate for 15 minutes before luminescence reading (ultrasensitive, 0.1 seconds per well) on a plate reader. For the HBGA blocking assay, the 50% blocking titer (BT50), defined as the titer at which the luminescence reading was 50% of the positive control, was determined for each sample. Samples with a BT50 of less than 20 were assigned a value of 10. BT50 values ​​were plotted as geometric means with 95% confidence intervals using Graph Pad Prism software.

[0202] Figures 3A-3E illustrate HBGA blocking titers of serum samples from animals vaccinated with the test vaccines. The vaccine groups tested are indicated on the x-axis, and the BT50 for each of the five different genotypes of norovirus is plotted. The dotted line at 20 indicates the limit of detection. A value of 10 indicates no blocking, even at the lowest dilution tested. Compared to the monovalent vaccine candidate, administration of the pentavalent vaccine induced comparable HBGA blocking titers specific for the five norovirus genotypes GI.1, GII.2, GII.3, GII.4, and GII.6.

[0203] Non-human primate (NHP) research This study was designed to test the immunogenicity of candidate norovirus vaccines in nonhuman primates. The animals tested were rhesus macaques housed at the National Institute for Research in New Iberia, Louisiana, USA. The test vaccine contained mRNA sequences encoding the VP1 proteins of five norovirus genotypes, GI.1, GII.2, GII.3, GII.4, and GII.6, formulated in LNP. Control animals were vaccinated with empty LNP.

[0204] Animals were immunized intramuscularly on the posterior right thigh with a total volume of 500 μL of each test vaccine at weeks 0 and 4. The candidate vaccines evaluated in this study are described above and outlined below in Table 3. Serum was collected from all animals.

[0205] [Table 9]

[0206] To test for the presence of antibodies capable of binding to norovirus-derived VLPs, 384-well ELISA plates were coated with 50 ng / well of one of the following VLPs: GI.1, GII.2, GII.3, GII.4, or GII.6 VLPs, corresponding to norovirus genotypes: GI.1, GII.2, GII.3, GII.4, and GII.6. During coating, a liquid handler prepared 10-point, 4-fold serial dilutions of serum in blocking buffer. After coating, the ELISA plate was washed, incubated with blocking buffer, emptied, and then incubated with samples from the serum dilution plate. After a 2-hour incubation, the ELISA plate was washed, and goat anti-human IgG (Fc fragment-specific)-HRP conjugate was added to each well. The plate was incubated for 1 hour, washed, and incubated with chemiluminescent substrate for 15 minutes before reading the luminescence (ultrasensitive, 0.1 seconds per well) on a plate reader. Interpolated endpoint titers were calculated as the highest dilution at which the relative luminescence (RLU) signal exceeded the 50,000 RLU cutoff. Samples whose dilutions did not exceed the threshold were given a titer of "25," and for samples that exceeded the threshold at the highest dilution tested—1:13,107,200—a value of 13,107,200 was used for the titer. Samples whose well value was exactly 50,000 RLU were given the interpolated titer value at that exact dilution. Serum antibody titers were plotted as geometric mean titers with 95% confidence intervals using GraphPad Prism software.

[0207] Figures 4A-4E illustrate interpolated endpoint titers of serum samples from animals vaccinated with the test vaccines. The vaccine groups tested are indicated on the x-axis, and binding to VLPs from each of five different norovirus genotypes is plotted. The dotted line at 50 indicates the limit of detection. A value of 25 indicates no binding, even at the lowest dilution tested. Vaccination with two dose levels of the pentavalent vaccine candidate, 50 μg or 200 μg, elicited comparable and robust serum IgG binding antibody titers specific for the five norovirus genotypes GI.1, GII.2, GII.3, GII.4, and GII.6. The pentavalent vaccine candidate and the monovalent GII.4 vaccine elicited comparable serum IgG ELISA titers against GII.4. A second administration of the pentavalent vaccine at both dose levels boosted antibody titers, which remained stable through week 8.

[0208] To explore functional antibody responses, we assessed the ability of serum to block VLP binding to HBGA. Depending on the test genotype, 384-well HBGA plates were coated with 250 ng / well of porcine gastric mucin (PGM) or 1:1000 diluted human saliva. During coating, a liquid handler prepared 10-point, 2-fold serial dilutions of serum in blocking buffer. After dilution, 30 μL / well of one of GI.1, GII.2, GII.3, GII.4, or GII.6 VLPs was added to each sample. After coating, the HBGA plates were washed, incubated with blocking buffer, emptied, and incubated with the mixture from the serum dilution + VLP plate. After a 2-hour incubation, the plates were washed, and rabbit serum hyperimmunized with the corresponding VLP was added to each well. After a 1-hour incubation, the plates were washed, and goat anti-rabbit IgG (Fc fragment-specific)-HRP was added. Plates were incubated for 1 hour, washed, and incubated with chemiluminescent substrate for 15 minutes before luminescence reading (ultrasensitive, 0.1 seconds per well) on a plate reader. For the HBGA blocking assay, the 50% blocking titer (BT50), defined as the titer at which the luminescence reading was 50% of the positive control, was determined for each sample. Samples with a BT50 of less than 20 were assigned a value of 10. BT50 values ​​were plotted as geometric means with 95% confidence intervals using Graph Pad Prism software.

[0209] Figures 5A-5E illustrate HBGA blocking titers of serum samples from animals vaccinated with the test vaccines. The vaccine groups tested are shown on the x-axis, and the BT50 for each of the five different norovirus genotypes is plotted. The dotted line at 20 indicates the limit of detection. A value of 10 indicates no blocking, even at the lowest dilution tested. Vaccination with two dose levels of the pentavalent vaccine candidate, 50 μg or 200 μg, elicited comparable and robust HBGA blocking antibody titers specific for the five norovirus genotypes GI.1, GII.2, GII.3, GII.4, and GII.6. The pentavalent vaccine candidate and the monovalent GII.4 vaccine elicited comparable HBGA blocking antibody titers against GII.4. A second dose at both dose levels boosted the antibody titers.

[0210] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the present disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0211] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the scope of the following claims.

Claims

1. A messenger ribonucleic acid (mRNA) polynucleotide encoding a Norovirus VP1 polypeptide, wherein said mRNA polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70% identity thereto, and wherein at least one uridine residue in said mRNA polynucleotide is replaced with N1-methylpseudouridine.

2. 2. The mRNA polynucleotide of claim 1, wherein the mRNA polynucleotide is derived from a Norovirus selected from the group consisting of GII.4, GI.1, GII.2, GII.3, and GII.6 Norovirus.

3. 3. The mRNA polynucleotide of claim 1 or 2, wherein the mRNA polynucleotide further comprises a heterologous 5' untranslated region (UTR) and a heterologous 3' UTR.

4. The mRNA polynucleotide of claim 3 , wherein the 5′UTR comprises the polynucleotide sequence of SEQ ID NO:

6.

5. 5. The mRNA polynucleotide of claim 4, wherein the 5'UTR further comprises a 5' cap structure N7-methyl-G(3'OMe)ppp.

6. The mRNA polynucleotide of any one of claims 3 to 5, wherein the 3'UTR comprises a poly(A) sequence and / or a poly(C) sequence.

7. 7. The mRNA polynucleotide of claim 6, wherein the poly(A) sequence comprises 10 to 200 adenosine nucleotides and / or the poly(C) sequence comprises 10 to 200 cytosine nucleotides.

8. The mRNA polynucleotide of claim 6, wherein the poly(A) sequence comprises 10 to 100 adenosine nucleotides.

9. The mRNA polynucleotide of claim 6, wherein the poly(A) sequence comprises 10 to 80 adenosine nucleotides.

10. The mRNA polynucleotide of claim 6, wherein the poly(A) sequence comprises 50 to 70 adenosine nucleotides.

11. The mRNA polynucleotide of claim 6, wherein the poly(C) sequence comprises 10 to 100 cytosine nucleotides.

12. The mRNA polynucleotide of claim 6, wherein the poly(C) sequence comprises 20 to 70 cytosine nucleotides.

13. The mRNA polynucleotide of claim 6, wherein the poly(C) sequence comprises 20 to 60 cytosine nucleotides.

14. The mRNA polynucleotide of claim 6, wherein the poly(C) sequence comprises 10 to 40 cytosine nucleotides.

15. 8. The mRNA polynucleotide of claim 7, wherein the poly(A) sequence comprises 80 adenine nucleotides.

16. The mRNA polynucleotide of any one of claims 3 to 9, wherein the 3'UTR comprises the polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:

8.

17. 11. The mRNA polynucleotide of claim 10, wherein the 3'UTR comprises the polynucleotide sequence of SEQ ID NO:

7.

18. The mRNA polynucleotide of claim 10, wherein the 3'UTR comprises the polynucleotide sequence of SEQ ID NO:

8.

19. The mRNA polynucleotide is sequenced in a 5' to 3' direction as follows: a 5'UTR comprising the polynucleotide sequence of SEQ ID NO:6; A polynucleotide encoding a Norovirus VP1 polypeptide, wherein the polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70% identity thereto; and 3'UTR comprising the polynucleotide sequence of SEQ ID NO:7 Including, 11. The mRNA polynucleotide of any one of claims 1 to 10, wherein the 5'UTR further comprises the 5' cap structure N7-methyl-G(3'OMe)ppp, and all uridine residues in the polynucleotide encoding the Norovirus VP1 polypeptide are replaced with N1-methylpseudouridine.

20. The mRNA polynucleotide is sequenced in a 5' to 3' direction as follows: a 5'UTR comprising the polynucleotide sequence of SEQ ID NO:6; A polynucleotide encoding a Norovirus VP1 polypeptide, wherein the polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70% identity thereto; and 3'UTR comprising the polynucleotide sequence of SEQ ID NO:8 Including, 11. The mRNA polynucleotide of any one of claims 1 to 10, wherein the 5'UTR further comprises the 5' cap structure N7-methyl-G(3'OMe)ppp, and all uridine residues in the polynucleotide encoding the Norovirus VP1 polypeptide are replaced with N1-methylpseudouridine.

21. 21. The mRNA polynucleotide of any one of claims 1 to 20, wherein the mRNA polynucleotide comprises at least one histone stem-loop structure.

22. 22. The mRNA polynucleotide of any one of claims 1 to 21, wherein at least 50% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine.

23. 22. The mRNA polynucleotide of any one of claims 1 to 21, wherein at least 75% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine.

24. 22. The mRNA polynucleotide of any one of claims 1 to 21, wherein at least 85% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine.

25. 22. The mRNA polynucleotide of any one of claims 1 to 21, wherein at least 95% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine.

26. 22. The mRNA polynucleotide of any one of claims 1 to 21, wherein 100% of the uridine residues in the mRNA polynucleotide are replaced with N1-methylpseudouridine.

27. A combination of one or more mRNA polynucleotides according to any one of claims 1 to 26.

28. A composition comprising at least one mRNA polynucleotide encoding the Norovirus VP1 polypeptide of any one of claims 1 to 26, and a pharmaceutically acceptable carrier.

29. A composition comprising at least two mRNA polynucleotides encoding the Norovirus VP1 polypeptide of any one of claims 1 to 26, and a pharmaceutically acceptable carrier.

30. A composition comprising at least three mRNA polynucleotides encoding the Norovirus VP1 polypeptide of any one of claims 1 to 26, and a pharmaceutically acceptable carrier.

31. A composition comprising at least four mRNA polynucleotides encoding the Norovirus VP1 polypeptide of any one of claims 1 to 26, and a pharmaceutically acceptable carrier.

32. A composition comprising at least five mRNA polynucleotides encoding the Norovirus VP1 polypeptide of any one of claims 1 to 26, and a pharmaceutically acceptable carrier.

33. 1. A composition comprising five mRNA polynucleotides encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier, wherein each of said five mRNA polynucleotides is, in the 5' to 3' direction: a 5'UTR comprising the polynucleotide sequence of SEQ ID NO:6; A polynucleotide encoding a Norovirus VP1 polypeptide, wherein the polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70% identity thereto; and 3'UTR comprising the polynucleotide sequence of SEQ ID NO:7 Including, wherein the 5'UTR further comprises the 5' cap structure N7-methyl-G(3'OMe)ppp, and wherein the composition comprises each of SEQ ID NOs: 1 to 5, and sequences having at least 70% identity thereto, and wherein in each of SEQ ID NOs: 1 to 5, and sequences having at least 70% identity thereto, all uridine residues in the five mRNA polynucleotides are replaced with N1-methylpseudouridine.

34. 1. A composition comprising five mRNA polynucleotides encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier, wherein each of said five mRNA polynucleotides is, in the 5' to 3' direction: a 5'UTR comprising the polynucleotide sequence of SEQ ID NO:6; A polynucleotide encoding a Norovirus VP1 polypeptide, wherein the polynucleotide is selected from the group consisting of SEQ ID NOs: 1-5 and sequences having at least 70% identity thereto; and 3'UTR comprising the polynucleotide sequence of SEQ ID NO:8 Including, wherein the 5'UTR further comprises the 5' cap structure N7-methyl-G(3'OMe)ppp, and wherein the composition comprises each of SEQ ID NOs: 1 to 5, and sequences having at least 70% identity thereto, and wherein in each of SEQ ID NOs: 1 to 5, and sequences having at least 70% identity thereto, all uridine residues in the five mRNA polynucleotides are replaced with N1-methylpseudouridine.

35. 35. The composition of any one of claims 28-34, wherein the composition further comprises one or more additional mRNA polynucleotides derived from a Norovirus selected from the group consisting of genotype group I Norovirus, genotype group II Norovirus, genotype group III Norovirus, genotype group IV Norovirus, and genotype group V Norovirus.

36. A composition comprising at least one mRNA polynucleotide encoding a Norovirus VP1 polypeptide and a pharmaceutically acceptable carrier.

37. 37. The composition of claim 36, further comprising at least one, at least two, at least three, at least four, or at least five mRNA polynucleotides encoding Norovirus VP1 polypeptides.

38. 38. The composition of claim 36 or claim 37, wherein the Norovirus VP1 polypeptide is derived from a genotype group I Norovirus or a genotype group II Norovirus.

39. 39. The composition of claim 38, wherein the Norovirus VP1 polypeptide is from a genotype group selected from the group consisting of GII.4, GI.1, GII.2, GII.3, and GII.6 Norovirus.

40. The composition of any one of claims 36 to 39, wherein the Norovirus VP1 polypeptide is selected from the group consisting of SEQ ID NOs: 14 to 18 and sequences having at least 70% identity thereto.

41. 41. The composition of any one of claims 28 to 40, wherein each mRNA polynucleotide is separately encapsulated in a lipid nanoparticle (LNP) comprising one or more cationic or polycationic compounds.

42. 41. The composition of any one of claims 28 to 40, wherein each mRNA polynucleotide is co-encapsulated in an LNP comprising one or more cationic or polycationic compounds.

43. 43. The composition of claim 41 or 42, wherein each mRNA polynucleotide encapsulated in the LNP is present in equal amounts.

44. The composition of claim 41 or 42, wherein each mRNA polynucleotide encapsulated in the LNP is not present in equal amounts.

45. 45. The composition of any one of claims 41 to 44, wherein the LNP comprises one or more of a cationic lipid, a sterol, a phospholipid, and a polyethylene glycol-lipid.

46. The composition of any one of claims 41 to 45, wherein the LNP comprises a cationic lipid, a sterol, a phospholipid, and a polyethylene glycol-lipid.

47. 47. The composition of any one of claims 41-46, wherein the LNPs comprise 34-59 mol% cationic lipid, 30-48 mol% sterol, 10-24 mol% phospholipid, and 1-2 mol% polyethylene glycol-lipid.

48. The cationic lipid is Table 1 The composition of any one of claims 41 to 47, selected from the group consisting of:

49. The sterol is 【Chemistry 1】 The composition according to any one of claims 41 to 48, wherein

50. The phospholipid is Table 2 The composition of any one of claims 41 to 49, selected from the group consisting of:

51. The polyethylene glycol-lipid Table 3 The composition of any one of claims 41 to 50, selected from the group consisting of:

52. 48. The composition of any one of claims 45 to 47, wherein the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

53. 48. The composition of any one of claims 45 to 47, wherein the sterol is cholesterol.

54. 48. The composition of any one of claims 45 to 47, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

55. 48. The composition of any one of claims 45 to 47, wherein the polyethylene glycol-lipid is dimyristoylglycerol-polyethylene glycol (DMG-PEG).

56. 56. The composition of any one of claims 45-55, wherein the LNPs comprise 49-59 mole % (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

57. 57. The composition of any one of claims 45-56, wherein the LNPs comprise about 58 mole % (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

58. 58. The composition of any one of claims 45-57, wherein the LNPs comprise about 49 mole % (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

59. A vaccine comprising an mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27 or a composition according to any one of claims 28 to 58.

60. 60. The vaccine of claim 59, wherein the vaccine further comprises a pharmaceutically acceptable carrier.

61. 61. The vaccine of claim 59 or 60, wherein the vaccine further comprises an adjuvant.

62. 62. The vaccine of any one of claims 59 to 61, wherein the vaccine is monovalent, bivalent, trivalent, tetravalent or pentavalent.

63. 63. The mRNA polynucleotide of any one of claims 1 to 26, the combination of claim 27, the composition of any one of claims 28 to 58 or the vaccine of any one of claims 59 to 62, wherein said mRNA polynucleotide, said combination, said composition and / or said vaccine is immunogenic in a mammalian species.

64. 63. A kit or kit-of-parts comprising an mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27, a composition according to any one of claims 28 to 58 or a vaccine according to any one of claims 59 to 62, wherein said kit or kit-of-parts may further comprise a liquid vehicle for solubilization and / or technical instructions providing information on the administration and dosage of the components.

65. An mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27, a composition according to any one of claims 28 to 58, or a vaccine according to any one of claims 59 to 62, or a kit or kit-of-parts according to claim 64, for use as a medicament.

66. 65. An mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27, a composition according to any one of claims 28 to 58, or a vaccine according to any one of claims 59 to 62, or a kit or kit-of-parts according to claim 64, for use in the manufacture of a medicament for the treatment or prevention of an infection by a Norovirus or a disorder associated with an infection by a Norovirus.

67. 65. An mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27, a composition according to any one of claims 28 to 58, or a vaccine according to any one of claims 59 to 62, or a kit or kit-of-parts according to claim 64, for use in the treatment or prevention of an infection by a Norovirus or a disorder associated with an infection by a Norovirus.

68. The mRNA polynucleotide of any one of claims 1 to 26, the combination of claim 27, the composition of any one of claims 28 to 58, or the vaccine of any one of claims 59 to 62, or the kit or kit of parts of claim 64, wherein an effective amount of the active components of said nucleic acid, said combination, said composition, said vaccine or said kit or kit-of-parts is administered by injection.

69. 64. A method for treating or preventing a disorder, said method comprising administering to a subject in need thereof an effective amount of an mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27, a composition according to any one of claims 28 to 58, or a vaccine according to any one of claims 59 to 62, or a kit or kit-of-parts according to claim 64.

70. 70. The method of claim 69, wherein the disorder is a Norovirus infection or a disorder associated with a Norovirus infection.

71. A vector comprising one or more nucleic acids encoding the mRNA polynucleotide of any one of claims 1 to 26.

72. A host cell comprising an mRNA polynucleotide according to any one of claims 1 to 26, a combination according to claim 27 or a vector according to claim 71.

73. A polypeptide encoded by the mRNA polynucleotide of any one of claims 1 to 26.

74. 10. A method for producing a virus-like particle (VLP) comprising one or more Norovirus VP1 polypeptides, the method comprising transcribing an mRNA polynucleotide of any one of claims 1 to 26, the combination of claim 27, or the vector of claim 71 in a recombinant nucleic acid expression system under conditions suitable for assembly of the expressed one or more Norovirus VP1 polypeptides into VLPs.

75. 75. A VLP comprising one or more Norovirus VP1 polypeptides produced according to the method of claim 74.

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