Disulfide-stabilized norovirus virus-like particle design

Abstract

Disclosed herein are synthetic VP1 proteins that contain engineered disulfide bonds. In some embodiments, the synthetic VP1 proteins with engineered disulfide bonds self-assemble into non-infectious virus-like particles (VLPs) that are more stable that non-infectious VLPs that resulted from self-assembly of VP1 proteins that lack engineered disulfide bonds.

Description

DISULFIDE-STABILIZED NOROVIRUS VIRUS-LIKE PARTICLE DESIGNCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 763,597, filed February 26, 2025, and 63 / 719,758, filed November 13, 2024, the contents of which are hereby incorporated by reference in their entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (26069-WO-PCT SL.xmh Size: 29.267 bytes, created on January 2, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] This disclosure relates generally to synthetic disulfide-stabilized virus-like particles (VLPs) and to synthetic norovirus VP1 proteins that contain engineered disulfide bonds.BACKGROUND

[0004] Norovirus, also referred to as “Norwalk virus,'’ was named after an outbreak of acute gastroenteritis in an elementary school in Norwalk, Ohio. Symptoms of norovirus infection appear within 12-48 hours of exposure and are characterized by nausea, vomiting, persistent diarrhea, lethargy, myasthenia, myalgia, headache, cough, and / or fever. Although severe illness is uncommon, and norovirus infections are generally self-limiting, norovirus remains one of the most common causes of acute gastroenteritis in people of all ages. There are approximate 700 million norovirus infections reported annually worldwide, 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 family Calicivlrldae . Noroviruses contain a linear, nonsegmented, positive-sense RNA genome of approximately 7.5 kilobases, encoding a large polyprotein which is cleaved into six smaller non-structural proteins (NS 1 / 2 to NS7) by the viral 3C-like protease (NS6), a major structural protein (VP1) of about 58-60 kDa and a minor capsid protein (VP2). Noroviruses can be classified genetically into at least seven different genogroups (GI, GII, Gill, GIV, GV. GVI. and GVII). which can be further divided into different genetic clusters or genotypes. Most noroviruses that infect humans belong to genogroups GI and GIL Noroviruses from genogroup II, genotype 4 (abbreviated as GII.4) account for the majority of adult outbreaks of gastroenteritis and often sweep across the globe.

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

[0007] At present, there is no specific prophylactic or therapeutic treatment for norovirus infections. Therapy is limited to palliative care of acute infections. There is therefore a strong need for additional therapeutic measures against norovirus infections.SUMMARY

[0008] The present disclosure relates to synthetic norovirus virus like particles (VLPs) that are more stable than their wild-type counterparts, due to engineered disulfide bonds in synthetic VP1 proteins. In some embodiments, the engineered disulfide bonds occur in the S domain of the synthetic VP 1 proteins.

[0009] In some embodiments, the synthetic VP1 proteins disclosed herein are a mutant VP1 proteins of a norovirus genogroup (GI, GII, Gill, GIV, GV, GVI, and GVII). In some embodiments, the norovirus genogroup is GII. In some embodiments, the synthetic VP1 proteins disclosed herein are mutant VP1 proteins of the norovirus genotype GII.3. In some embodiments, the synthetic VP1 proteins disclosed herein are mutant VP1 proteins of norovirus genotype GII.4. In some embodiments, the synthetic VP1 proteins disclosed herein are mutant VP1 proteins of norovirus genotype GII.6.

[0010] In an aspect, VP1 proteins are disclosed that are capable of self-assembly into non-infectious virus-like particles (VLPs). In some embodiments, self-assembly of the VLPs comprising engineered disulfide bonds in the VP1 proteins results in increased yield of said VLPs relative to VLPs that result from self-assembly of VP1 proteins that do not contain the engineered disulfide bonds. In some embodiments, self-assembly of the VLPs comprising engineered disulfide bonds in the VP1 proteins results in VLPs with higher thermal stability relative to the VLPs that result from self-assembly of VP1 proteins that do not contain the engineered disulfide bonds. In some embodiments, self-assembly of the VLPs comprising engineered disulfide bonds in the VP1 proteins results in VLPs that are more compact relative to VLPs that result from self-assembly of VP1 proteins that do not contain the engineered disulfide bonds. In some embodiments, self-assembly of the VLPs comprising engineered disulfide bonds in the VP1 proteins results in VLPs that are less poly-disperse relative to VLPs that result from self-assembly of VP1 proteins that do not contain the engineered disulfide bonds.

[0011] In an aspect, synthetic polynucleotides are disclosed that encode synthetic VP1 polypeptides. In some embodiments, the synthetic polynucleotides encode synthetic VP1 polypeptides that contain engineered disulfide bonds. In some embodiments, the polynucleotides are DNA. In some embodiments, the polynucleotides are RNA. In some embodiments, the RNA is a messenger ribonucleic acid (mRNA).

[0012] In an aspect, vaccines are disclosed that comprise polynucleotides. In some embodiments, the polynucleotides encode VP1 proteins that contain engineered disulfide bonds. In some embodiments, the polynucleotides are deoxyribonucleic acids (DNA). In some embodiments, the polynucleotides are ribonucleic acids (RNA). In some embodiments, the RNA is messenger ribonucleic acid (mRNA).

[0013] In an aspect, a synthetic polypeptide is provided, comprising a synthetic VP1 polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of the amino acid residues at positions 112 and 189 is a cysteine. In some embodiments, the VP1 polypeptide comprises any one of SEQ ID NOs: 1, 7, and 11.

[0014] In an aspect, a synthetic polynucleotide is provided, comprising a nucleic acid sequence that encodes a synthetic VP1 polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of the amino acid residues at positions 112 and 189 is a cysteine. In some embodiments, the synthetic polynucleotide encodes a VP1 polypeptide comprising the polypeptide sequence of any one of SEQ ID NOs: 1 , 7, and 11.

[0015] In an aspect, a synthetic polynucleotide is provided, comprising a DNA nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3, wherein the nucleic acid sequence encodes a synthetic VP1 polypeptide wherein at least one of the amino acid residues at positions 112 and 189 is a cysteine. In some embodiments, the synthetic polynucleotide comprises SEQ ID NO: 3. In some embodiments, the nucleic acid is DNA.

[0016] In an aspect, a synthetic polypeptide is provided, comprising a mutated VP1 polypeptide comprising a sequence of amino acids as set forth in any one of SEQ ID NOs: 2, 8. and 12, with the exception of a single amino acid substitution relative to the amino acid sequence in any of the foregoing sequences wherein one of (i) amino acid position 112 and (ii) amino acid position 189, in any of SEQ ID NOs: 2, 8, and 12, comprises a cysteine residue.

[0017] In an aspect, a composition is provided comprising any of the synthetic polypeptides described herein and a pharmaceutically acceptable carrier. In some embodiments, the synthetic polypeptide comprises a sequence that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions112 and 189 of SEQ ID NOs: 1, 7, and 11 is a cysteine. In some embodiments, the synthetic polypeptide comprises any one of SEQ ID NOs: 1, 7, and 11.

[0018] In an aspect, a synthetic polynucleotide is provided comprising a DNA sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 3, 9, and 13, wherein the nucleic acid sequence encodes any of the VP1 polypeptide sequences described herein. In some embodiments, the DNA sequence comprises any one of SEQ ID NOs: 3, 9, and 13.

[0019] In an aspect, a synthetic polynucleotide is provided, comprising a synthetic messenger ribonucleic acid (mRNA) sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 5, wherein the mRNA encodes a synthetic VP1 polypeptide. In some embodiments, the synthetic mRNA sequence comprises SEQ ID NO: 5.

[0020] In an aspect, a composition is provided, comprising any synthetic polynucleotide disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the synthetic polynucleotide comprises a sequence that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 3, 9, and 13. In some embodiments, the synthetic polynucleotide comprises any one of SEQ ID NOs: 3, 9 and 13.

[0021] In some embodiments, the synthetic polynucleotide comprises a sequence that is at least 90% identical or at least 95% identical to SEQ ID NO: 5. In some embodiments, the synthetic polynucleotide comprises SEQ ID NO: 5.

[0022] In an aspect, a composition is provided, comprising any synthetic polypeptide disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the synthetic polypeptide comprises a sequence that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions 112 and 189 of SEQ ID NOs: 1, 7, and 11 is a cysteine. In some embodiments, the synthetic polypeptide comprises any one of SEQ ID NOs: 1, 7, and 11.

[0023] In an aspect, a composition is provided comprising any of the synthetic polynucleotides comprising mRNA described herein, wherein the synthetic polynucleotide is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA comprises a sequence that is at least 90% or at least 95% identical to SEQ ID NO: 5.

[0024] In an aspect, a vector is provided comprising a polynucleotide that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 3, 9, and 13. In some embodiments, the polynucleotide sequence comprises any one of SEQ ID NOs: 3, 9. and 13.

[0025] In an aspect, a non-infectious virus-like particle (VLP) is provided comprising a polypeptide that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions 112 and 189 of SEQ ID26069NOs: 1, 7, and 11 is a cysteine. In some embodiments, the polypeptide comprises any one of SEQ ID NOs: 1, 7, and 11.

[0026] In an aspect, a host cell is provided comprising (i) a synthetic polypeptide that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 1. 7, and 11, (ii) a synthetic polynucleotide that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 3, 5, 9, and 13, (iii) anon-infectious virus-like particle (VLP) comprising a synthetic polypeptide that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: I, 7, and 11 or (iv) a composition comprising a synthetic polynucleotide that is at least 90% identical or at least 95% identical to SEQ ID NO: 5 and a lipid nanoparticle (LNP). In some embodiments, the synthetic polypeptide in (i) comprises any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the synthetic polynucleotide in (ii) comprises any one of SEQ ID NOs: 3, 5, 9, and 13. In some embodiments, the synthetic polypeptide in (iii) comprises any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the synthetic polynucleotide in (iv) comprises any one of SEQ ID NOs: 5, 9, and 13.

[0027] In an aspect, a method of treating or preventing a disorder related to a norovirus infection is provided, comprising administering to a subject in need thereof an effective amount of any of synthetic polypeptides, synthetic polynucleotides, or compositions described herein.

[0028] In an aspect, a method of making GII.3 non-infectious virus-like particles (VLPs) is provided comprising: expressing a synthetic polypeptide that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11 in an expression system under conditions suitable for assembly into non-infectious VLPs. In some embodiments, the synthetic polypeptide comprises any one of SEQ ID NOs: 1. 7, and 11.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGs. 1 A and IB provide data showing VP1 expression from transiently transfected Expi293 cells, as described in Example 2. Clarified supernatant (1A) and lysate (IB) fractions were separated via SDS-PAGE and either stained with SimplyBlue Safestain or blotted onto membranes and probed with an anti-VPl antibody. The position of full length VP1 is denoted by black arrows. The positions of cleaved fragments of strains GII.3 and GII.6 are denoted by grey arrows. Marker proteins in kilodalton (kDa) are indicated.

[0030] FIGs. 2A-2E provide data showing that the DS1 mutation stabilizes GII.3 VLPs, as described in Example 3 and Example 4. FIG. 2F shows that the overall yield of VLPs is higher for GII.3 DS1 compared to GII.3 WT, as described in Example 3 and Example 4.

[0031] FIGs. 3A-3D provide image analysis showing that after a single freeze thaw cycle, the DS1 mutation results in uniform VLPs, as describe in Example 5.

[0032] FIGs. 4A-4D provide image analysis showing that, without freezing, the DS1 mutation results in uniform VLPs. as described in Example 5.

[0033] FIGs. 5 A and 5B provide data showing binding data of GII.3 DS1 and GII.3 WT, to human saliva samples, as described in in Example 6.

[0034] FIGs. 6A and 6B provide data showing ELISA binding curves of GII.3 DS1 and GII.3 WT, under different conditions, as described in Example 6.

[0035] FIGs. 7A-7C provides antibody responses elicited by mRNAs encoding GII.3 DS1 or GII.3 WT in mice, as described in Example 8.

[0036] FIG. 8 provides data showing binding data of GII.3 DS1 and GII.3 WT, to monoclonal antibodies.

[0037] FIGs. 9A and 9B provides data showing mouse immunogenicity data of mRNAs encoding either GII.3 or GII.3-DS1, using assay reagent GII.3 VLP.DETAILED DESCRIPTION

[0038] The present disclosure relates to synthetic norovirus VP1 proteins that contain engineered disulfide bonds. In some embodiments, the engineered disulfide bonds are in the shell (S) domain of the norovirus VP1 proteins.

[0039] In some embodiments, the invention relates to norovirus VP1 proteins of genotype GII.3 or GII.6 of the GII genogroup of norovirus, wherein the VP1 proteins comprise engineered disulfide bonds.

[0040] In some embodiments, the synthetic norovirus VP1 proteins disclosed herein selfassemble into non-infectious virus-like particles (VLPs). In some embodiments, production of the non-infectious VLPs from self-assembly of the synthetic VP1 proteins disclosed herein result in increased VLP yield relative to VLP yield that is produced through self-assembly of wild-type VP1 proteins. In some embodiments, production of the non-infectious VLPs from self-assembly of the synthetic VP1 proteins disclosed herein result in higher thermal stability of the VLPs relative to VLPs that are produced through self-assembly of wild-type VP1 proteins. In some embodiments, production of non-infectious VLPs from self-assembly of the synthetic VP1 proteins disclosed herein result in more uniform and less poly disperse VLPs relative to VLPs that are produced through self-assembly of wild-ty pe VP1 proteins.

[0041] In some embodiments, the synthetic norovirus VP1 proteins that are capable of selfassembly into to non-infectious VLPs comprises a sequence that is at least 90% identical, at least93% identical, at least 95% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions 112 and 189 of SEQ ID NOs: 1, 7, and 11 is a cysteine. In some embodiments, the synthetic VP1 proteins comprise any one of SEQ ID NOs: 1. 7, and 11.

[0042] In some embodiments, disclosed herein are synthetic polynucleotides that encode synthetic VP1 proteins that contain engineered disulfide bonds. In some embodiments, the polynucleotides comprise DNA. In some embodiments, the DNA sequence comprises a sequence that is at least 90% identical, at least 93% identical, at least 95% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 3, 9. and 13. wherein the polynucleotide encodes a synthetic norovirus VP1 polypeptide having one or more engineered disulfide bonds, as described herein. In some embodiments, the DNA sequence comprises any one of SEQ ID NOs: 3, 9, and 13.

[0043] In some embodiments, the polynucleotides comprise RNA. In some embodiments, the RNA sequence comprises an mRNA sequence. In some embodiments, the mRNA sequence comprises a sequence that is at least 90% identical, at least 93% identical, at least 95% identical, at least 98% identical, or at least 99% identical, to SEQ ID NO: 5, wherein the polynucleotide encodes a synthetic norovirus VP1 polypeptide having one or more engineered disulfide bonds, as described herein. In some embodiments, the mRNA sequence comprises SEQ ID NO: 5.

[0044] In some embodiments disclosed herein are vaccines that comprise an mRNA sequence. In some embodiments, the mRNA sequence comprises a sequence that is at least 90% identical, at least 93% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 5, wherein the mRNA sequence encodes a synthetic norovirus VP1 polypeptide having one or more engineered disulfide bonds, as described herein. In some embodiments, the mRNA sequence comprises SEQ ID NO: 5. In some embodiments, the mRNA sequence comprises SEQ ID NO: 5. In some embodiments, the vaccine comprises a pharmaceutically acceptable carrier. In some embodiments, the vaccine comprises an adjuvant.

[0045] In some embodiments, disclosed herein are vectors that comprise polynucleotides. In some embodiments, the polynucleotides comprise a sequence that is at least 90% identical, at least 93% identical, at least 95% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 3, 9, and 13, wherein polynucleotides encode a VP1 polypeptide as described herein (i.e., a VP1 polypeptide having a cysteine at one or both of amino acid residues 112 and 189). In some embodiments, the polynucleotide comprises any one of SEQ ID NOs: 3, 9, and 13.

[0046] In some embodiments, disclosed herein are non-infectious VLPs that comprise synthetic polypeptides. In some embodiments, the synthetic polypeptides comprise a sequence that is at least 90%, at least 93% at least 95%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions 112 and 189 of SEQ ID NOs: 1, 7, and 11 is a cysteine. In some embodiments, the sequence comprises any one of SEQ ID NOs: 1, 7, and 11.

[0047] In some embodiments, disclosed herein are norovirus vaccines that comprise any mRNA sequence disclosed herein. In some embodiments, the mRNA sequence comprises a sequence that is at least 90% identical, at least 93% identical, at least 95% identical, at least 98% identical, or at least 99% to SEQ ID NO: 5, wherein the mRNA encodes a polypeptide encoding a VP1 protein having a cysteine at one or both of the amino acid residues at positions 112 and 189. In some embodiments, the mRNA sequence comprises SEQ ID NO: 5.DEFINITIONS

[0048] Listed herein are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0049] Unless defined otherwise, 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 used herein and the laboratory7procedures in cell culture, molecular genetics, organic chemistry7, and peptide chemistry are those well-known and commonly employed in the art.

[0050] 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” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0051] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0052] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg"’ is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0053] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s).” “having,” “has.” “may.”“contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0054] The terms “% identical'’, “% identity” or similar terms are intended to refer, in particular to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared and introducing gaps, if necessary , to achieve maximum percent sequence identity. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. Two sequences can be optimally aligned for comparison even when the sequences are of different lengths, which includes optimally aligning sequences in which one sequence is truncated on either or both of its 5' and 3’ ends relative to the sequence to which it is being aligned. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology7algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity' search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0055] As used herein, the terms “GII.3 DS1”, “GII.4 DS1”, and “GII.6 DS1” refer to mutated VP1 proteins of the respective norovirus genotypes or mutated polynucleotides of respective norovirus genotypes that encode mutated VP1 proteins. The mutated VP1 proteins contain cysteine residues at amino acid positions 112 and 189. The mutated polynucleotides encode VP1 proteins that contain cysteine residues at amino acid positions 112 and 189.

[0056] As used herein, the terms “GII.3 WT”, GII.4 WT”, and “GII.6 WT” refer to a wild-type VP1 proteins of a norovirus of the respective norovirus genotypes or wild-type polynucleotides of respective norovirus genotypes that encode wild-type VP1 proteins.

[0057] As used herein, the term "DS " refers to a VP 1 protein that has the following mutations: N112C and G189C. Thus, for example, the phrase “GII.3 DS refers to a GII.3 genotype that has N112C and G189C mutations in its VP1 protein.

[0058] As used herein, reference to the amino acid positions 112 and 189, refer to these amino acid positions on a VP 1 protein as they correspond to the WT VP1 amino acid sequences of GII.3 WT (SEQ ID NO: 2), GII.6 WT (SEQ ID NO: 8), and GII.4 WT (SEQ ID NO: 12). Also, as used herein, reference to N112C and G189C is referring to mutations at amino acid positions 112 and 189 as these amino acid positions correspond to the WT amino acid sequences of GII.3 WT (SEQ ID NO: 2). GII.6 WT (SEQ ID NO: 8), and GII.4 WT (SEQ ID NO: 12).Proteins / polypeptides and VP1 proteins

[0059] The term ‘‘protein” in its broadest sense, includes any compound and / or substance that comprise a polymer of amino acids. The polymers are referred to as polypeptides. In some embodiments, the polypeptides disclosed herein comprise VP1 proteins.

[0060] VP1 proteins are viral proteins and are the main structural component of the viral capsid of certain viruses, including norovirus. VP1 proteins contain different structural components, including the N-terminus (N), the C-terminus (C). and the shell (S) domain. The S domain functions in assembly of the capsid shell of norovirus.

[0061] In some embodiments, the VP1 proteins described herein derive from norovirus. In some embodiments, the VP1 proteins described herein derive from any genotype of the norovirus. In some embodiments, the VP1 proteins derive from the GII.3 genotype of norovirus. In some embodiments, the VP1 proteins disclosed herein are mutated forms of VP1 proteins that derive from the GII.3 genotype of norovirus.

[0062] In some embodiments, disclosed herein are synthetic VP1 proteins. In some embodiments, the synthetic VP1 proteins contain engineered disulfide bonds. In some embodiments, the engineered disulfide bonds are within the shell domain of the VP1 proteins. In some embodiments, the engineered disulfide bonds within the shell domain function create a more stable capsid shell of norovirus after self-assembly of the VP1, relative to a capsid shell proteins that are assembled from VP1 proteins that lack engineered disulfide bonds.

[0063] In some embodiments, the engineered disulfide bonds result from mutating wild type VP1 protein residues to increase the number of cysteine residues. In some embodiments, the mutated VP1 proteins disclosed herein contain between one and ten additional cysteine residues relative to a wild-type VP1 protein, for example, one additional cysteine residue, two additional cysteine residues, three additional cysteine residues, four additional cysteine residues, fiveadditional cysteine residues, six additional cysteine residues, seven additional cysteine residues, eight additional cysteine residues, nine additional cysteine residues, or ten additional cysteine residues.

[0064] In an aspect, disclosed herein is a VP 1 protein that has been mutated. In some embodiments, the VP1 protein that is mutated derives from the GII.3 norovirus. In some embodiments, the VP1 protein comprises SEQ ID NO: 2, with the exception that the sequence is mutated such that it comprises one or two additional cysteine residues relative to the non-mutated form. In some embodiments. SEQ ID NO: 2 is mutated such that any one or more of amino acid positions 112 and 189 comprises a cysteine residue. In some embodiments. SEQ ID NO: 2 is mutated such that both amino acid positions 112 and 189 comprise a cysteine residue.

[0065] In an aspect, a synthetic VP1 protein is provided that contains one or two additional cysteine residues relative to a wild-tjpe VP1 protein of the GII.3 norovirus. In some embodiments, the synthetic VP1 polypeptide comprises a sequence that is at least 90% identical or at least 95% identical to any of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the sequence is about 90%, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7. and 11. wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the sequence comprises any one of SEQ ID NOs: 1, 7, and 11.

[0066] In some embodiments, the synthetic VP1 polypeptide comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1. 7, and 11, wherein both of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the synthetic VP1 polypeptide sequence comprises a sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein both of amino acid positions 112 and 189 comprise a cysteine residue.Virus-Like Particles (VLPs)

[0067] Virus-like particles (VLPs) are particles that have a similar structure to particles of viruses, but that lack genetic material. In some embodiments, the VLPs described herein are characterized as non-infectious VLPs.26069

[0068] Non-infectious VLPs contain an outer shell known as a capsid. The main component of the capsid is a VP1 protein, for example a norovirus VP1 protein. The capsid is created through self-assembly of the VP1 proteins, which results in an icosahedral capsid structure. In some embodiments, the synthetic VP1 proteins described herein comprise engineered disulfide bonds. In some embodiments, the engineered disulfide bonds are in the shell (S) domain of the synthetic VP1 proteins.

[0069] In some embodiments, self-assembly of the synthetic VP1 proteins that contain engineered disulfide bonds result in more stable capsid shells and more stable non-infectious VLPs, relative to capsid shells and non-infectious VLPs that result from self-assembly of VP1 proteins that lack engineered disulfide bonds. In some embodiments, self-assembly of the synthetic VP1 proteins that contain engineered disulfide bonds result in a higher yield of non-infectious VLPs, relative the yield of non-infectious VLPs that result from self-assembly of VP1 proteins that lack engineered disulfide bonds. In some embodiments, self-assembly of the synthetic VP1 proteins that contain engineered disulfide bonds result in non-infectious VLPs that have a higher thermal stability, relative to non-infectious VLPs that result from self-assembly of VP1 proteins that lack engineered disulfide bonds. In some embodiments, self-assembly of the synthetic VP1 proteins that contain engineered disulfide bonds result in non-infectious VLPs that are more compact and less polydisperse, relative to non-infectious VLPs that result from self-assembly of VP1 proteins that lack engineered disulfide bonds.

[0070] In an aspect, a non-infectious VLP is provided comprising any one of the VP1 proteins described herein. In some embodiments, the VLP comprises VP1 protein comprising a sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 1, 7. and 11. wherein at least one of amino acids 112 and 189 comprise a cysteine residue. In some embodiments, the VP1 protein comprises a sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein both of amino acids 112 and 189 comprise cysteine residues. In some embodiments, the sequence comprises any one of SEQ ID NOs: 1. 7, and 11.

[0071] In some embodiments, the VP1 protein comprises a sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 2, 8, and 12 wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the VP1 protein comprises a sequence that is at least 90% identical or at least 95% identical to any of SEQ ID NOs: 2, 8, and 12 and both of amino acid positions 112 and 189 comprise a cysteine residue.

[0072] In some embodiments, any of the synthetic VP1 polypeptides described herein are capable of self-assembly into non-infectious VLPs. In some embodiments, the VP1 polypeptides capable of self-assembly into non-infectious VLPs comprise a sequence that is at least 90%26069identical or at least 95% identical to any one of SEQ ID NOs: 1, 1, and 11, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the sequence is about 90%, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the sequence comprises any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the synthetic VP1 polypeptides that are capable of self-assembly in non-infectious VLPs, comprise sequence that is sequence is about 90%, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the synthetic VP1 polypeptides that are capable of self-assembly in non-infectious VLPs, comprise sequence that is sequence is about 90%, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein both of amino acid positions 112 and 189 comprise a cysteine residue.NUCLEIC ACIDS / POLYNUCLEOTIDES

[0073] The term ‘‘nucleic acid,” in its broadest sense, includes any compound and / or substance that comprises a polymer of nucleotides. These polymers are referred to as “polynucleotides.” Nucleic acids (also referred to as polynucleotides) may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TN As), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a 0- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), or chimeras or combinations thereof.

[0074] In some embodiments, polynucleotides of the present disclosure comprise synthetic DNA sequences. In some embodiments, the synthetic DNA sequences encode for synthetic VP1 proteins. In some embodiments, the synthetic DNA sequences encode for any synthetic VP1 protein disclosed herein.

[0075] In some embodiments, disclosed herein is a synthetic DNA sequence that includes a sequence that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 3, 9, and 13. In some embodiments, the DNA sequence comprises a sequence that is about 90%26069identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 3, 9, and 13, wherein the DNA sequence encodes a VP1 polypeptide, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the DNA sequence comprises any one of SEQ ID NOs: 3, 9, and 13.

[0076] In some embodiments, vectors are disclosed herein that comprise DNA sequences that encode for a synthetic VP1 protein sequence. In some embodiments, the vector comprises a DNA sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 3, 9. and 13, wherein the DNA sequence encodes a VP1 protein having a cysteine residue at amino acid positions 112 and / or 189. In some embodiments, the DNA sequence comprises a sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 3, 9, and 13, wherein the DNA sequence encodes a VP1 protein having an amino acid residue at position 112 and / or 189. In some embodiments, the DNA sequence comprises any one of SEQ ID NOs: 3, 9, and 13.

[0077] In some embodiments, the DNA sequences disclosed herein encode a synthetic VP1 protein sequence that is at least 90% identical or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the DNA sequence encodes for a synthetic VP1 protein sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the DNA sequence encodes for a synthetic VP1 protein sequence in which the synthetic VP1 protein sequence comprises any one of SEQ ID NOs: 1. 7, and 11.

[0078] In some embodiments, polynucleotides of the present disclosure comprise messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (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.

[0079] In some embodiments, polynucleotides of the present disclosure comprise synthetic mRNA. In some embodiments, the synthetic mRNA sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein wherein at least26069one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the mRNA sequence comprises a sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the synthetic mRNA sequence comprises SEQ ID NO: 5.

[0080] The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA, the 'T"s would be substituted for ‘‘U”s.

[0081] 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. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and / or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

[0082] In some embodiments, disclosed herein is an mRNA sequence that encodes for a synthetic VP1 protein sequence in which the synthetic VP1 protein sequence is at least 90% identical to SEQ ID NO: 1, wherein the mRNA encodes a VP1 protein wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the mRNA sequence encodes for a synthetic VP1 protein sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 1, wherein the mRNA encodes a VP1 protein wherein at least one of amino acid positions 112 and 189 comprise a cysteine residue. In some embodiments, the mRNA sequence encodes for a synthetic VP1 protein sequence in which the synthetic VP1 protein sequence comprises SEQ ID NO: 1.RNA Vaccines

[0083] In some embodiments, one or more of the RNA disclosed herein are part of a vaccine.

[0084] In some embodiments, an RNA polynucleotide of a norovirus vaccine encodes 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, an RNA polynucleotide of a norovirus vaccine encodes antigenic polypeptides derived from the GII.3 strain of norovirus.26069

[0085] In some embodiments, the RNA vaccine comprises a synthetic mRNA sequence that is at least 90% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein wherein at least one of amino acid positions 112 and 189 of the VP1 protein comprises a cysteine residue. In some embodiments, the RNA vaccine comprises a sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein wherein at least one of amino acid positions 112 and 189 of the VP1 protein comprises a cysteine. In some embodiments, the RNA vaccine comprises a synthetic mRNA comprising SEQ ID NO: 5.

[0086] In some embodiments, disclosed herein is an RNA vaccine that comprises an mRNA sequence that encodes for a synthetic VP1 protein sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 of the VP1 protein comprises a cysteine. In some embodiments, the mRNA sequence of the RNA vaccine encodes a synthetic VP1 protein sequence that is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1. 7, and 11, wherein at least one of amino acid positions 112 and 189 comprises a cysteine. In some embodiments, the mRNA sequence of the RNA vaccine encodes a synthetic VP 1 protein sequence comprising any one of SEQ ID NOs: 1 , 7, and 11.

[0087] In addition to a coding region, a mature mRNA comprises a 5' untranslated region (UTR) and a 3' UTR, which play important roles in regulating gene expression. A “5' UTR” refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (z.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. A “3' UTR” refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

[0088] The 5' UTR may further comprise a 5' cap sequence. 5' capping of polynucleotides may be completed concomitantly during an in vztz'o-transcription reaction using the following chemical RNA cap analogs to generate the 5' guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5’)ppp(5') G [the ARCA cap]:G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5,)ppp(5')G (New England BioLabs, Ipswich, MA). 5' capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-26069transferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes are preferably derived from a recombinant source. In certain embodiments, the 5' cap structure is N7-Methyl-G(3’OMe)ppp.

[0089] The 3' UTR may further comprise a poly(A) sequence or a poly(C) sequence, which are typically located at the 3’ end of an mRNA. A poly(A) sequence, also called poly(A) tail or 3' poly(A) tail, is typically understood to be a sequence of adenosine nucleotides.

[0090] In some embodiments, a poly(A) sequence may be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.Moreover, poly(A) sequences, or poly(A) tails may be generated in vitro by enzymatic polyadenylation of the RNA, e.g., using Poly(A)polymerases (PAP) derived from / • / . coli or yeast. In addition, poly adenylation of RNA can be achieved by using immobilized PAP enzymes e.g., in a polyadenylation reactor. See International Patent Application Publication WO 2016 / 174271.Chemical Modifications

[0091] In an aspect, RNA vaccines are provided that comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one norovirus antigenic polypeptide. In some embodiments, the RNA comprises at least one chemical modification.

[0092] The terms “chemical modification’’ and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxy ribonucleosides in at least one of their position, pattern, percent, or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5' terminal mRNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered “modified” if they contain amino acid substitutions, insertions or a combination of substitutions and insertions. In some embodiments, the mRNA sequences of SEQ ID NOs: 5 and 6 are modified through substitutions, insertions, or a combination of substitutions and insertions.

[0093] In some embodiments, a particular region of a polynucleotide contains one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g.. a modified mRNA polynucleotide (e.g., one or more26069modifications to SEQ ID NOs: 5 or 6)), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative 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 in the cell or organism, respectively (e.g., a reduced innate response).

[0094] Modifications of polynucleotides include, without limitation, modifications to polynucleotides described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprise modifications that are naturally -occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an intemucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).

[0095] Polynucleotides (e.g.. RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an intemucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.

[0096] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides). A “nucleoside'’ refers to a compound containing a sugar molecule (e.g.. a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or nonnatural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0097] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding26069between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker may be incorporated into polynucleotides of the present disclosure.

[0098] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that are useful in the vaccines of the present disclosure include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; l,2'-O-dimethyladenosine; 1 -methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-O-methyladenosine; 2'-0-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-hydroxynorvalylcarbamoyladenosinc; N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; Nl-methyl-adenosine; N6. N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-Amino-2’-deoxy-ATP; 2’-Azido-2’-deoxy-ATP; 2'-Deoxy-2'-a-aminoadenosine TP: 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine: aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine: 7-deaza-8-aza-adenosme; 7-methyladenine; 1 -Deazaadenosine TP; 2’Fluoro-N6-Bz-deoxyadenosine TP; 2’-OMe-2-Amino-ATP; 2’O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP; 2-aminoadenine; 2- Aminoadenosine TP; 2- Amino- ATP; 2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'-difluoroadenosine TP; 2'-Deoxy-2'-a-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-26069fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine TP; 2'-Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine 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 '-Homo-adenosine 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-amin op urine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine; 2-(thio)cytosine; 2’-Amino-2’-deoxy-CTP; 2’-Azido-2’-deoxy-CTP; 2'-Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2-0-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cylosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5 -(trifluoromethyl (cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-l-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-l-methyl-l-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’Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2’-O-Methyl-N4-Acetyl-cytidine TP; 2’0-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy-2'-a-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-(l-propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(l-Propynyl)ara-cytidine26069TP; 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'-Homo-cytidine TP; 5 -Methoxy cytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine; l,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1 -methylguanosine; a-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2’-Amino-2’-deoxy-GTP; 2’-Azido-2’-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl )guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1 -methyl-6-thio-guanosine; 6-methoxy -guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2’Fluoro-N2-isobutyl-guanosine TP; 2’O-methyl-N2-isobutyl-guanosine TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP; 2'-b-Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine 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'-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1 -methylinosine; Inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 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; 1-26069methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; 1 -methylpseudouridine; 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-pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxy carbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyacetic acid- Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; Nl-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)- 2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil; a-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-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; l-(aminoalkylarnino-carbonylethylenyl)-2-(thio)-pseudouracil; 1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; l-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 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' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-l-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalky l)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-26069methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2.4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dial ky laminoalky I (uraci I ; 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-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5 -iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; P seudo-UTP-l-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1 -methyl- 1-deaza-pseudouri dine; 1-propynyl-uridine; 1-taurinomethyl-l-methyl-uridine; l-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-l -methylpseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudo uridine; (±)l-(2-Hydroxypropyl)pseudouridine TP; (2R)-l-(2-Hydroxypropyl)pseudouridine TP; (2S)-l-(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; l-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; l-(2,2-Di ethoxy ethyl)pseudouridine TP; l-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1 -(2,4,6-Trimethyl-benzyl)pseudo-UTP; l-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1 -(2- Aminoscarboxy ethyl)pseudo-UTP; l-(2-Amino-ethyl)pseudo-UTP; l-(2-Hydroxyethyl)pseudouridine TP; 1 -(2 -Methoxy ethyl)pseudouridine TP; l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; l-(3,4-Dimethoxybenzyl)pseudouridine TP; l-(3-Amino-3-carboxypropyl)pseudo-UTP; l-(3-Amino-propyl)pseudo-UTP; l-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1 -(4-Amino-4-carboxybutyl)pseudo-UTP; l-(4-Amino-benzyl)pseudo-UTP; l-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP; 1 -(4-Azidobenzyl)pseudouridine TP; l-(4-Bromobenzy I (pseudouridine TP; l-(4-Chlorobenzyl)pseudouridine TP; l-(4-26069Fluorobenzyl)pseudouridine TP; 1 -(4-Iodobenzyl)pseudouridine TP; l-(4-Methanesulfonylbenzyl)pseudouridine TP; 1 -(4-Methoxybenzyl)pseudouridine TP; l-(4-Methoxy-benzyl)pseudo-UTP; l-(4-Methoxy-phenyl)pseudo-UTP; 1 -(4-Methylbenzyl)pseudouridine TP; l-(4-Methyl-benzyl)pseudo-UTP; l-(4-Nitrobenzyl)pseudouridine TP; l-(4-Nitro-benzyl)pseudo-UTP; l(4-Nitro-phenyl)pseudo-UTP; l-(4-Thiomethoxybenzyl)pseudouridine TP; l-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethylbenzyl)pseudouridine TP; l-(5-Amino-pentyl)pseudo-UTP; l-(6-Amino-hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; 1- j3-|2-(2-Aminoethoxy)-ethoxy ]-propionyl } pseudouridine TP; 1-Acetylpseudouridine TP; l-Alkyl-6-(l-propynyl)-pseudo-UTP; l-Alkyl-6-(2-propynyl)-pseudo-UTP; l-Alkyl-6-allyl-pseudo-UTP; l-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP; l-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo-UTP; 1 -Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2 -pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1 -Cyanomethylpseudouridine TP; 1-Cyclobutj Imethyl-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-iso-propyl-pseudo-UTP; l-Me-2-thio-pseudo-UTP; l-Me-4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1 -Methanesulfonylmethylpseudouridine TP; 1 -Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; l-Methyl-6-(4-morpholino)-pseudo-UTP; 1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; l-Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; l-Methyl-6-azido-pseudo-UTP; l-Methyl-6-bromo-pseudo-UTP; l-Methyl-6-butyl-pseudo-UTP; l-Methyl-6-chloro-pseudo-UTP; l-Methyl-6-cyano-pseudo-UTP; l-Methyl-6-dimethylamino-pseudo-UTP; l-Methyl-6-ethoxy-pseudo-UTP; l-Methyl-6-ethylcarboxylate-pseudo-UTP; l-Methyl-6-ethyl-pseudo-UTP; l-Methyl-6-fluoro-pseudo-UTP; 1 -Methyl-6-formyl-pseudo-UTP; 1 -Methyl-6-hydroxy amino-pseudo-UTP; 1 -Methyl-6-hy droxy-pseudo-UTP; l-Methyl-6-iodo-pseudo-UTP; l-Methyl-6-iso-propyl-pseudo-UTP; l-Methyl-6-methoxy-pseudo-UTP; l-Methyl-6-methylamino-pseudo-UTP; l-Methyl-6-phenyl-pseudo-UTP; l-Methyl-6-propyl-pseudo-UTP; l-Methyl-6-tert-butyl-pseudo-UTP; l-Methyl-6-trifluoromethoxy-pseudo-UTP; 1 -Methyl-6-trifluoromethyl-pseudo-UTP; 1 -Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-26069Pivaloylpseudouridine 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-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2’ -anhydro-uridine TP; 2’-bromo-deoxyuridine TP; 2’-F-5-Methyl-2’-deoxy-UTP; 2’-OMe-5-Me-UTP; 2’-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O-Methyl-5-(l-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(l-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-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1 -(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP l-[3-(2-ethoxy)]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2-[2- {2(2-ethoxy)-ethoxy} -ethoxy] -ethoxy)-ethoxy}] propionic acid; Pseudouridine TP l-[3-{2-(2-[2-ethoxy ] -ethoxy )-ethoxy}] propionic acid; Pseudouridine TP l-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-Nl-3-propionic acid; Pseudo-UTP-Nl-4-butanoic acid; Pseudo-UTP-Nl-5-pentanoic acid; Pseudo-UTP-Nl-6-hexanoic acid; Pseudo-UTP-Nl-7-heptanoic acid; Pseudo-UTP-Nl-methyl-p-benzoic acid; Pseudo-UTP-Nl-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodifiedhydroxy wybutosine; 4-demethylwyosine; 2,6-(diamino)purine; l-(aza)-2-(thio)-3-(aza)-phenoxazin-1 -yl: 1 ,3-(diaza)-2-(oxo)-phenthiazin-l-yl; 1 ,3-(diaza)-2-(oxo)-phenoxazin-l -yl; l,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2‘ methyl, 2’amino, 2’azido, 2'fluro-cytidine; 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-pyridopyrimidine-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-on-3-yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin4-yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)-l.3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl; 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin4-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7-(guanidiniumalkylhy droxy )- 1 -(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(guanidiniumalkylhy droxy)- 1 ,3-(diaza)-2-(oxo)-phenoxazin-l -yl; 7-(guanidiniumalkyl-hydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl; 7-(guanidiniumalkylhy droxy )-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine;Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines; O-alkylated derivative; ortho-(aminoalkylhy droxy )-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl;Tubercidine; 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.

[0099] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0100] In some embodiments, modified nucleobases in polynucleotides (e.g.. RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (\| / ), N1 -methylpseudouridine (mh| / ). 2-thiouridine, 4 ’-thiouridine, 5-methylcy tosine, 2-thio- 1 -methyl- 1-deaza-pseudouri dine, 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-l -methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5 -methoxy uridine and 2’-O-methyl uridine. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0101] In some embodiments, one or more polynucleotide of the invention is modified by replacing one or more uridine residue with one or more modified nucleobase. In certain embodiments, at least 50% of the uridine residues of the mRNA polynucleotide are replaced with N1 -methylpseudouridine. In certain embodiments, between 50% and 55%, between 55% and 60%, between 60% and 65%, between 65% and 70%, between 70% and 75%, between 75% and 80%, 80% and 85%, between 85% and 90%, between 90% and 95%, and between 95% and 100% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 90% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 91% of the uridine residues of the mRNA polynucleotide are replaced with N1 -methylpsuedouridine. In certain embodiments, 92% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 93% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 94% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 95% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 96% of the uridine residues of the mRNApolynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 97% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 98% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridine. In certain embodiments, 99% of the uridine residues of the mRNA polynucleotide are replaced with Nl-methylpsuedouridineln certain embodiments, 100% of the uridine residues of the mRNA polynucleotide are replaced with N 1 -methylpsuedouridine.

[0102] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of 1-methyl-pseudouridine (m )- 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (\| / ), a-thio-guanosine and a-thio-adenosine. In some embodiments, the polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0103] In some embodiments, polynucleotides (e.g.. RNA polynucleotides, such as mRNA polynucleotides) comprise pseudouridine (y) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (ml\| / ). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (ml\| / ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g.. RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine (s2U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g.. RNA polynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine (mo5U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2’-O-methyl uridine. In some embodiments polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2’-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0104] In some embodiments, polynucleotides (e g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly26069modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0105] Exemplary nucleobases and nucleosides having a modified cytosine 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.

[0106] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified cytosine, or, a modified uridine include 5-cyano uridine, and 4’-thio uridine.

[0107] In some embodiments, a modified nucleobase is a modified adenine. Exemplary' nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1 -methyladenosine (mlA), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0108] In some embodiments, a modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methyl-inosine (mH), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m7G), 1 -methylguanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0109] The polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide, or in a given predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly(A) tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+LJ, A+C, G+U, G+C. U+C. A+G+U. A+G+C. G+U+C or A+G+C.

[0110] The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%. from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to2606980%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.[OHl] The polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, 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, the polynucleotides may contain a modified pyrimidine such as a 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 uracil in the polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having 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 cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).IN VITRO TRANSCRIPTION (IVT) OF RNA (E G., MRNA)

[0112] “In vitro transcription (IVT)’’ is a method to synthesize mRNA from a linear DNA template using an RNA polymerase, where the RNA polymerase binds to a promoter sequence and transcribes mRNA through the addition of complimentary nucleotides in the 5' to 3' direction. Norovirus vaccines of the present disclosure comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA) (e.g., SEQ ID NO: 5 and SEQ ID NO: 6, and modified forms thereof). mRNA, for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.” In some embodiments, the at least one RNA polynucleotide has at least one chemical modification. The at least one chemical modification may include, but is expressly not limited to, any modification described herein.

[0113] Linear DNA templates encoding for mRNA sequences are prepared from purified plasmid DNA and may contain an RNA polymerase promoter sequence, encoded 5' UTR, coding sequence, encoded 3' UTR with a poly (A) tail, and a linearization cut site. Plasmid DNA sequences are generated using standard cloning methods in a plasmid vector backbone, such as26069pUC19, pUC57, or pmRNAxp, and plasmid DNA is purified from fermentation of transformed E. coli strains typically used for plasmid production, such as Stable or DH5a, using standard methods (See, e.g., US Patent Application Publication No. 2019 / 0083602, US Patent Application Publication No. 2020 / 0392518). mRNA may be produced in an IVT reaction with a co-transcriptional cap analog addition and a DNA template encoded poly(A) tail, or the cap and / or poly(A) tail may be added enzymatically after the IVT reaction. DNA plasmids may contain an adenine-guanine (AG) sequence at the start of the 5' UTR after a T7 promoter sequence to enable co-transcriptional capping with a Cap 1 AG analog (m7G(5')ppp(5')(2’OMeA)pG). DNA plasmids may also contain a specific restriction enzyme cut site after an encoded 3' poly (A) tail. Plasmids may be linearized in a digestion reaction containing purified plasmid DNA at a concentration of about 0.1-2 mg / mL and the appropriate restriction enzyme, such as BbsI or BspQI, at a concentration of about 500-20,000 U / mg in a digest mixture that may contain tris hydrochloride, magnesium, potassium, acetate, albumen, 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 min, preferably 60 min.

[0114] The linear DNA template may be purified by solvent extraction, alcohol precipitation, centrifugation, chromatographic, and / or filtration-based methods to remove the uncut plasmid DNA, restriction enzyme, and digest mixture components. Purified linear DNA templates may be prepared at a concentration of about 0.5-2 mg / mL, preferably 1 mg / mL, in water or a buffer containing tris hydrochloride, ethylenediaminetetraacetic acid (EDTA), and / or other excipient at a solution pH of about 7-8.

[0115] The IVT reaction is performed in a temperature-controlled reaction vessel with an incubation temperature range of about 34-40°C, preferably 37°C. The linear DNA template may be added to the IVT reaction mixture at a concentration range of about 10-200 pg / mL, preferably 40-60 pg / mL. Natural nucleoside triphosphates (NTPs) including adenosine triphosphate (ATP), guanosine triphosphate (GTP). cytidine triphosphate (CTP), uridine triphosphate (UTP), or modified NTPs, such as pseudouridine (*P), Nhmethylpseudouridine (mlP), N6-methyladenosine (m6A), or N5-methylcytidine (m5C), may be used in the IVT reaction, preferably ATP, GTP, CTP, and mlvP. The NTPs may be incorporated in mRNA as a mixture of natural and substituted modified NTPs, such as a mixture of 50% UTP and 50% mlxP. Preferably, 100% mllP is added to fully substitute for UTP. The NTPs may be added to the IVT reaction mixture at a concentration range of about 2-15 mM, or preferably 8-14 mM for ATP, GTP, CTP and 4-7 mM for mlv. 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), may be added to26069the IVT reaction mixture at a concentration range of about 1-10 rnM, preferably 3-5 mM. An RNA polymerase, such as T7 RNA polymerase, may be added to the IVT reaction mixture at a concentration range of about 2,000-20,000 U / mL, preferably 8,000-12,000 U / mL.

[0116] A pyrophosphatase, such as yeast inorganic pyrophosphatase, and RNase inhibitor, such as murine RNase inhibitor, may be added to the IVT reaction mixture at a concentration range of 1-5 U / mL and 500-2,000 U / mL, respectively. Other components of the IVT reaction mixture may include tris hydrochloride, magnesium, spermidine, dithiothreitol (DTT), sodium, chloride, potassium, acetate, phosphate, polysorbate-20, polysorbate-80, TritonX-100, glycerol and / or other excipients at a final solution pH of about 6-9. The IVT reaction mixture is typically incubated with mixing for 1-6 hr, preferably 3-4 hr. Preferably, the mRNA is produced in the IVT reaction with a co-transcriptional 5’ Cap 1 AG analog and a DNA template encoded 3’ poly(A) tail and no additional enz matic capping or poly(A) tail addition reactions are necessary . The IVT reaction is terminated by addition of a DNase, such as DNase I, to digest the linear DNA template into small oligonucleotides and stop transcription. DNase is added to the IVT reaction mixture at a concentration range of about 100-500 U / mL, preferably 350 U / mL, along with calcium chloride at a concentration range of about 1-5 mM, preferably 3.5 mM, and incubated at about 34-40°C, preferably 37°C for 0.5-3 hr, preferably 1 hr. After DNase digestion, a proteinase, such as Proteinase K, may be added to the IVT reaction mixture to digest enzymes used in the IVT reaction into small peptides. Proteinase is added to the IVT reaction mixture at a concentration range of about 0.05-0.2 mg / mL, preferably 0.1 mg / mL, along with sodium dodecyl sulfate (SDS) and additional DTT, and incubated at about 34-40°C, preferably 37°C for 0.5-3 hr, preferably 1 hr. Preferably, only DNase treatment is performed, and no proteinase treatment is performed. After DNase or DNase followed by proteinase treatments are complete, the IVT reaction mixture is adjusted to 10-100 mM EDTA, preferably 50 mM to quench enzy me activity.

[0117] 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.

[0118] An initial tangential flow filtration (TFF) step may be performed after DNase and proteinase treatment for buffer exchange and clearance of small impurities. TFF is operated by pumping a feed solution across a membrane at a transmembrane pressure (TMP) of about 1-10 psi, where solution components larger than the molecular weight cut off (MWCO) remain in the26069retentate, and components smaller than the MWCO may be permeated through the membrane. The membrane MWCO may range from 30-500 kDa in a flat sheet or hollow fiber format and may be composed of a variety of materials including poly ethersulfone (PES), polysulfone (PS), or regenerated cellulose (RC). When the IVT reaction mixture is processed across TFF, the large mRNA molecules are retained while digested impurities and other small molecules are cleared in the permeate. Prior to starting TFF, the IVT reaction mixture may be diluted 2-20-fold in water or a buffer containing tris hydrochloride, sodium phosphate, or sodium citrate at a concentration of about 1-100 mM, preferably 10 mM tris hydrochloride, at a pH of about 6-8 and EDTA at a concentration of about 1-10 mM, preferably 2 mM. A TFF membrane loaded with approximately 1 -25 g mRNA per m2of membrane area of diluted IVT reaction mixture operated at a TMP of about 3-7 psi and crossflow shear rate of about 2000-12000 s'1, may be used to buffer exchange the diluted IVT mixture across 5-15 diafiltration volumes (DVs) into water or a buffer containing tris hydrochloride, 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. Preferably, a 50 kDa hollow fiber PES membrane loaded with 5-10 g-mRNA / m2operated at a TMP of about 4 psi and a crossflow shear rate of 4000 s'1is used to buffer exchange the diluted IVT mixture across 10 DVs into 10 mM tris hydrochloride, 2 mM EDTA, pH 7.2.

[0119] The purified mRNA-containing solution in the TFF retentate may be forwarded to hybridization affinity chromatography media using an oligo deoxythymine (oligo dT) ligand conjugated to a stationary phase to selectively bind mRNA through complimentary base pairing of the oligo dT ligand with the mRNA poly(A) tail. IVT reaction contaminants including RNA polymerase, pyrophosphatase, RNase inhibitor, DNase, and proteinase, small molecules including free nucleotides, pyrophosphate, magnesium, spermidine, other IVT matrix excipients, and RNA fragments lacking a poly(A) tail are not expected to bind to the oligo dT ligand.Preferably, the DNase treated IVT reaction mixture is forwarded directly to oligo dT affinity chromatography, omitting the Proteinase K and initial TFF steps. The oligo dT ligand may consist of about 15-30 deoxythymine bases connected to a linker coupling the ligand to a support surface, such as a microporous polymethacrylate monolith, crosslinked poly(styrene-divinylbenzene) bead, or electrospun cellulose nanofibers. The mRNA-containing solution forwarded to the oligo dT hybridization affinity chromatography step may be diluted about 2-50-fold into an oligo dT binding matrix consisting of a salt, such as sodium chloride, potassium chloride, lithium chloride, guanidine hydrochloride, or other similar salts, at a concentration of about 100-1000 mM, a buffer, such as tris hydrochloride, sodium phosphate, or sodium citrate, at a concentration of 5-100 mM at a pH of about 6-8, and EDTA at a concentration of about 1-1026069mM. Preferably, the DNase treated IVT mixture is diluted about 12-fold into an oligo dT binding matrix consisting of 400 mM sodium chloride, 10 mM tris hydrochloride, 2 mM EDTA, pH 7.2. The mRNA-containing solution may be pumped through oligo dT chromatography media with an approximate loading of about 1-8 mg / mL-media at a residence time of about 0.1-10 min. and upon binding of mRNA with a poly(A) tail, the column is washed with 2-10 column volumes (CV) of a mobile phase consisting of a salt, such as sodium chloride, potassium chloride, lithium chloride, guanidine hydrochloride, or other similar salts, at a concentration of about 10-200 mM, a buffer, such as tris hydrochloride, sodium phosphate, or sodium citrate, at a concentration of 5-100 mM at a pH of about 6-8, and EDTA at a concentration of about 1-10 mM. Preferably, the mRNA containing mixture is pumped through a microporous polymethacrylate monolith media at loading of 2-3 mg / mL-media and a residence time of 0.5 min, and the column is washed with 5 CVs of a buffer consisting of 50 mM sodium chloride, 10 mM tris hydrochloride, 2 mM EDTA, pH 7.2. The bound mRNA is eluted from the oligo dT ligand with 2-10 CVs of a low ionic strength mobile phase consisting of water or a buffer, such as tris hydrochloride, sodium phosphate, or sodium citrate, at a concentration of 1-20 mM at a pH of about 6-8. Preferably, the bound mRNA is eluted with 4-5 CVs of 10 mM tris hydrochloride, pH 7.2. The oligo dT chromatography media may be regenerated with sodium hydroxide and re-used for subsequent chromatography purification cycles. Preferably, a polymethacrylate monolith is be regenerated wi th 0.5 M sodium hydroxide and reused through about 2-6 purification cycles of the same DNase treated IVT mixture diluted in oligo dT binding buffer.

[0120] The mRNA-containing oligo dT hybridization affinity chromatography elution fraction may be purified by polishing chromatographic methods 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 polishing chromatographic methods may include mixed-mode chromatography, hydrophobic interaction chromatography, anion-exchange chromatography, reversed-phase chromatography, ceramic hydroxyapatite chromatography, sizeexclusion chromatography, or cellulose chromatography. Preferably, no additional polishing purification methods are performed after oligo dT chromatography.

[0121] The mRNA-containing oligo dT hybridization affinity7chromatography elution fraction is buffer exchanged and purified by a final TFF that may be operated similarly to the previously described initial TFF. The final TFF membrane may be operated at aTMP of 1-10 psi using a 30-500 kDa MWCO membrane in a flat sheet or hollow fiber format and may be composed polyethersulfone (PES), polysulfone (PS), or regenerated cellulose (RC). A 50-100 kDa hollow fiber PES membrane loaded with approximately 1-20 g mRNA per m2of membrane area26069operated at a TMP of 3-7 psi and crossflow shear rate of about 2000-12000 s'1may be used to concentrate the oligo dT elution fraction approximately 2-20-fold followed by a buffer exchange across 4-15 DVs into water or a buffer consisting of about 1-10 mM tris hydrochloride, sodium phosphate, or sodium citrate at a pH of about 5-8. Preferably, a 50 kDa hollow fiber PES membrane loaded with 5-10 g-mRNA / m2operated at a TMP of about 4 psi and a crossflow shear rate of 4000 s'1is used to concentrate 8-fold followed by a buffer exchange of 6 DVs into 1 mM sodium citrate, pH 6.4.

[0122] The mRNA-containing retentate from the final TFF is filtered at a flux of about 25-1000 L / m2-hr and a loading of about 10-1000 g / m2through a bioburden reduction filter with a nominal pore size of about 0.2 pm. The filter may be composed of a variety of materials including hydrophilic polyvinylidene fluoride (PVDF), PES, or cellulose acetate and may contain a prefilter with a nominal pore size ranging from about 0.2-1 pm. Preferably, the final TFF retentate is pumped at a flux of 300 L / m2-hr and a loading of 100 g / m2through a 0.2 pm PVDF filter. The concentration of mRNA in the bioburden reduction filter product is calculated from a sample measurement of absorbance at 260 nm using a spectrophotometer. The filtration product may be diluted with the final TFF diafiltration buffer to a target a final concentration of mRNA ranging from about 0.5-5 mg / mL. Preferably, the filtration product is diluted with 1 rnM sodium citrate, pH 6.4 to a mRNA concentration of about 1 mg / mL. The purified mRNA may be stored at refrigerated conditions at about 2-8°C or frozen at about -20°C or <-60°C.ANTIGENIC POLYPEPTIDES

[0123] An "‘antigenic polypeptide” is a polypeptide which induces an immune response when administered to an animal, preferably a mammal. In some embodiments, an antigenic polypeptide includes 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 polypeptide. In certain, embodiments, the norovirus VP1 polypeptide derives from the norovirus Gil.3 strain, the norovirus GII.4 strain, or the norovirus GII.6 strain. In certain embodiments, the antigenic polypeptide comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 7, and 11, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of amino acid positions 112 and 189 of the VP1 protein comprises a cysteine. In some embodiments, the antigenic polypeptide comprises any one of SEQ ID NOs: 1, 7, and 11.26069

[0124] Antigenic polypeptides of the present disclosure may be prepared by using any suitable method known in the art, including synthetic methods such as e.g., solid phase synthesis, as well as recombinant and in vitro methods, such as in vitro transcription reactions. In certain embodiments, antigenic polypeptides may be prepared by in vitro transcription of the polynucleotides disclosed herein.

[0125] A “variant” is a molecule that differs in its amino or nucleic acid sequence relative to a native sequence or a reference sequence. Sequence variants may possess substitutions, deletions, insertions, or a combination of any two or three of the foregoing, at certain positions within the amino or nucleic acid sequence, as compared to a native sequence or a reference sequence. Ordinarily, variants possess at least 50% identity to a native sequence or a reference sequence. In some embodiments, variants share at least 80% identity or at least 90% identity' with a native sequence or a reference sequence.

[0126] The present disclosure provides several types of compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion, and covalent variants and derivatives. The term “derivative” is synonymous with the term “variant” and generally refers to a molecule that has been modified and / or changed in any way relative to a reference molecule or a starting molecule. As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions, and / or additions, deletions, and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this disclosure.

[0127] “Substitutional variants” when referring to polynucleotides, are those that have at least one nucleotide residue in a native or starting sequence removed and a different nucleotide inserted in its place at the same position. In some embodiments, one or more uridine residues in an mRNA polynucleotide of the present disclosure are replaced with a pseudouridine. The modification of mRNA nucleosides, e g., with pseudouridine or a pseudouridine derivative, have been described previously. See International Patent Application Publication WO 2007 / 024708.

[0128] As used herein the terms “termini” or “terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions.

[0129] Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term “identity ,” as known in the26069art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity7also means the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity7of related peptides can be readily calculated by know n methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to 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 of the BLAST suite. See Stephen F. Altschul, et al. (1997)." Gapped BLAST and PSI-BLAST: anew generation of protein database search programs," Nucleic Acids Res. 25:3389-3402. Another popular local alignment technique is based on the Smith- Waterman algorithm. See Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197. A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm. See Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453. More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.LIPID NANOPARTICLES (LNPS)

[0130] 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, liposomes or vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single; unilamellar or26069multiple; multilamellar), or, in other embodiments, wherein the lipids coat an interior comprising a prophylactic or therapeutic product, or lipid aggregates or micelles, wherein the lipid encapsulated prophylactic or therapeutic product is contained within a relatively disordered lipid mixture. In certain embodiments, mRNA polynucleotides disclosed herein may be encapsulated together in a LNP comprising one or more cationic or poly cationic compounds. In some embodiments, the mRNA encapsulated in the LNP comprises a sequence that is at least 90% identical to SEQ ID NO: 5, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP 1 protein having a cysteine at amino acid positions 112 and / or 189. In some embodiments, the mRNA encapsulated in the LNP comprises SEQ ID NO: 5.

[0131] Except where noted, the LNP does not need to have an antigenic polypeptide incorporated therein and may be used to deliver a product (i.e. , mRNA polynucleotides) to a mammal when the LNP and mRNA polynucleotides in the same formulation.

[0132] As used herein, "polyamine” means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.

[0133] Unless otherw ise specified, mole % refers to a mole percent of total lipids.

[0134] In some embodiments, the LNPs of the compositions are composed of one or more cationic lipids (including ionizable cationic lipids) and one or more poly(ethyleneglycol)-lipids (PEG-lipids). In certain embodiments, the LNPs further comprise one or more non-cationic lipids. The one or more non-cationic lipids can include a phospholipid, phospholipid derivative, a sterol, a fatty acid, or a combination thereof.

[0135] Cationic lipids and ionizable cationic lipids suitable for the 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, rendering them positively charged at acidic pH but close to charge-neutral at physiologic pH. Cationic lipids are characterized by monovalent or multivalent cationic charge on their headgroups, which renders them positively charged at neutral pH. In certain embodiments, the cationic and ionizable lipid is capable of complexing with hydrophilic bioactive molecules to produce a hydrophobic complex that partitions into the organic phase of a two-phase aqueous / organic system. It is contemplated that both monovalent and polyvalent cationic lipids may be utilized to form hydrophobic complexes with bioactive molecules.

[0136] Preferred cationic and ionizable cationic lipids for use in forming the LNPs include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-26069(2,3dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride (“DOTMA”); N,NdistearylN,N-dimethylammonium bromide (“DDAB”); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylamntonium chloride (“DODAP”); 1,2 bis (oleoyloxy )-3-(trimethylammonio) propane (DOTAP); 3-(N-(N,N-dimethylaminoethane)-carbam-oyl)cholesterol (‘DC-Chol”); diheptadecylamidoglycylspermidine (“DHGS”) and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hy doxy ethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic lipids, as well as other components, are available which can be used. These include, for example, LIPOFECTIN® (commercially available cationic lipid nanoparticles comprising DOTMA and l,2dioleoyl-sn-3-phosphoethanolamine (“DOPE”), from GIBCOBRL, Grand Island. N.Y.. USA); and LIPOFECTAMINE® (commercially available cationic lipid nanoparticles comprising N-(l-(2,3dioleyloxy)propyl)N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA') and (“DOPE”), from (GIBCOBRL). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA. 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9, 12-dienyl-[l,3]dioxolan-4-ylmethyl)-dimethylamine, DLinKDMA (WO 2009 / 132131), DLin-K-C2-DMA (WO2010 / 042877), DLin-M-C3-DMA (WO2010 / 146740 and / or W02010 / 105209), DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate; Jayaraman et al., 2012, Angew. Chem. Int. Ed. Engl. 51:8529-8533). 2-{4-[(3P)-cholest-5-en-3-yloxy]butoxy[-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienlyloxyl]propan-l -amine) (CLinDMA), and the like. Other cationic lipids suitable for use include, e.g., the cationic lipids described in U.S. Pat. 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 include, e.g., Lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages 112 - 139) of International Patent Application Publication No. WO2011 / 076807 (which also discloses methods of making, and methods of using these cationic lipids).

[0137] In some embodiments, 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. W02010 / 021865.

[0138] In some embodiments, LNPs comprise one or more ionizable cationic lipids described in International Patent Application Publication No. WO2011 / 022460, or any pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds or salts therein.

[0139] When structures of the same constitution differ in respect to the spatial arrangement of certain atoms or groups, they are stereoisomers, and the considerations that are significant in analyzing their interrelationships are topological. If the relationship between two stereoisomers is26069that of an object and its nonsuperimposable mirror image, the two structures are enantiomeric, 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 one another. Cis-trans isomers differ only in the positions of atoms relative to a specified plane in cases where these atoms are, or are considered as if they were, parts of a rigid structure.Conformational isomers are isomers that can be interconverted by rotations about formally single bonds. Examples of such conformational isomers include cyclohexane conformations with chair and boat conformers, carbohydrates, linear alkane conformations with staggered, eclipsed and gauche conformers, etc. See J. Org. Chem. 35, 2849 (1970).

[0140] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that 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 one another. A mixture of enantiomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the Formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the Formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0141] In some embodiments, when the compounds contain one chiral center, the compounds exist in two enantiomeric forms and include both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixtures. The enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes which may be separated, for example, by26069crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate 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.

[0142] In some embodiments, designation of a specific absolute configuration at a chiral carbon of the compounds disclosed herein is understood to mean that the designated enantiomeric form of the compounds is in enantiomeric excess (ee) or in other words is substantially free from the other enantiomer. For example, the "R" forms of the compounds are substantially free from the "S" forms of the compounds and are, thus, in enantiomeric excess of the "S" forms. Conversely, "S" forms of the compounds are substantially free of "R" forms of the compounds and are, thus, in enantiomeric excess of the "R" forms. Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%. In a particular embodiment when a specific absolute configuration is designated, the enantiomeric excess of depicted compounds is at least about 90%.

[0143] In some embodiments, when a compound has two or more chiral carbons it 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 4 optical isomers and 2 pairs of enantiomers ((S,S) / (R,R) and (R,S) / (S,R)). The pairs of enantiomers (e.g., (S,S) / (R,R)) are mirror image stereoisomers of one another. The stereoisomers that are not mirror-images (e.g., (S,S) and (R,S)) are diastereomers. The diastereoisomeric pairs may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. In some embodiments, the compounds disclosed herein include each diastereoisomer of such compounds and mixtures thereof.

[0144] The LNPs may also comprise any combination of two or more of the cationic lipids described herein. In certain aspects, the cationic lipid typically comprises from about 0.1 to about 99.9 mole % of the total lipid present in said particle. In certain aspects, the cationic lipid can comprise from about 80 to about 99.9% mole %. In other aspects, the cationic lipid comprises from about 2% to about 70%, from about 5% to about 50%, from about 10% to about 45%, from about 20% to about 99.8%, from about 30% to about 70%, from about 34% to about 59%, from26069about 20% to about 40%, or from about 30% to about 40% (mole %) of the total lipid present in said particle.

[0145] The LNPs described herein can further comprise a noncationic lipid, which can be any of a variety of neutral uncharged, zwitterionic or anionic lipids capable of producing a stable complex. They are preferably neutral, although they can be negatively charged. Examples of noncationic lipids include phospholipid-related materials, such as natural phospholipids, synthetic phospholipid derivatives, fatty' acids, sterols, and combinations thereof. Natural phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI). Phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacyl-phosphatidylcholine (lyso PC), l-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:l), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0 and lethicin.

[0146] In certain embodiments, the non-cationic lipid is selected from: lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylet-hanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal). Noncationic lipids also include sterols such as cholesterol, stigmasterol or stigmastanol. Cholesterol is known in the art. See U.S. Patent Application Publication Nos: U.S. 2006 / 0240554 and U.S. 2008 / 0020058. In certain embodiments, the LNP comprise a combination of a phospholipid and a sterol.

[0147] Where present, the non-cationic lipid ty pically comprises from about 0.1% to about 65%. about 2% to about 65%, about 10% to about 65%. or about 25% to about 65% expressed as mole percent of the total lipid present in the LNP. The LNPs described herein further include a polyethyleneglycol (PEG) lipid conjugate (“PEG-lipid”) which may aid as a bilayer stabilizing component. The lipid component of the PEG lipid may be any non-cationic lipid described above26069including natural phospholipids, synthetic phospholipid derivatives, fatty acids, sterols, and combinations thereof. In certain embodiments, the PEG-lipids include, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., International Patent Application Publication No. WO 05 / 026372, PEG coupled to diacylglycerol (PEG-DAG) as described in. e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689; PEG coupled to phosphatidylethanolamine (PE) (PEG-PE), or PEG conjugated to 1 ,2-Di-O-hexadecyl-sn-glyceride (PEG-DSG), or any mixture thereof. See, e.g., U.S. Pat. No. 5,885,613.

[0148] In one embodiment, the PEG-DAG conjugate is a dilaurylglycerol (C 12)-PEG conjugate, a PEG dimyristylglycerol (C14)conjugate, a PEG-dipalmitoylglycerol (C16) conjugate, a PEG-dilaurylglycamide (C12) conjugate, a PEG-dimynstylglycamide (C14) conjugate, a PEG-dipalmitoylglycamide (Cl 6) conjugate, or a PEG-disterylglycamide (Cl 8). Those of skill in the art will readily appreciate that other diacylglycerols can be used in the PEGDAG conjugates.

[0149] In certain embodiments, PEG-lipids include, but are not limited to, PEG-dimyristolglycerol (PEG-DMG), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG- 1 ,2-dimyristyloxlpropy 1-3 -amine (PEG-c-DMA).

[0150] In certain embodiments, the PEG-lipid is PEG coupled to dimyristoylglycerol (PEG-DMG), e.g., as described in Abrams et al., 2010, Molecular Therapy 18(1): 171, and U.S. Patent Application Publication Nos. US 2006 / 0240554 and US 2008 / 0020058.

[0151] In certain embodiments, the PEG-lipid, such as a PEG-DAG, PEG-cholesterol, PEG-DMB, comprises a polyethylene glycol having an average molecular weight ranging of about 500 daltons to about 10,000 daltons, of about 750 daltons to about 5,000 daltons, of about 1,000 daltons to about 5,000 daltons, of about 1,500 daltons to about 3,000 daltons or of about 2,000 daltons. In certain embodiments, the PEG-lipid comprises PEG400, PEG1500, PEG2000 or PEG5000.

[0152] The acyl groups in any of the lipids described above are preferably acyl groups derived from fatty7acids having about CIO to about C24 carbon chains. In one embodiment, the acyl group is lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

[0153] The PEG-lipid conjugate typically comprises from about 0.1% to about 15%, from about 0.5% to about 20%, from about 1.5% to about 18%, from about 4% to about 15%, from about 5% to about 12%, from about 1% to about 4%, or about 2% expressed as a mole % of the total lipid present in said particle.26069

[0154] In certain embodiments, the LNPs comprise one or more cationic lipids, cholesterol and 1.2-Dimyristoyl-sn-glycerol methoxypoly ethylene glycol (PEG-DMG).

[0155] In certain embodiments, 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).

[0156] In some embodiments, the LNP comprises 34-59 mole % of a cationic lipid, 30-48 mole % of a sterol, 10-24 mole % of a phospholipid, and 1-2 mole % of a PEG-lipid.

[0157] In some embodiments, the cationic lipid is selected from the group consisting of:

[0158] In some embodiments, the sterol is:26069

[0159] In some embodiments, wherein the phospholipid is selected from the group consisting of:

[0160] In some embodiments, the polyethyleneglycol-lipid is selected from the group consisting of26069

[0161] In certain embodiments, the LNPs comprise lipid compounds assembled within the following molar ratios:26069Cationic Lipid (20-99.8 mole %)Non-cationic lipid (0.1-65 mole %) andPEG-DMG (0.1-20 mole %).

[0162] In certain embodiments, the LNPs comprise lipid compounds assembled within the following molar ratios:Cationic Lipid (30-70 mole %)Non-cationic lipid (20-65 mole %) andPEG-DMG (1-15 mole %).

[0163] In certain aspects of this embodiment, the non-cationic lipid is cholesterol. Exemplary LNPs may include cationic lipid / cholesterol / PEG-DMG at about the following molar ratios: 58 / 30 / 10.

[0164] In certain aspects of this embodiment, the non-cationic lipid is cholesterol and DSPC.Exemplary LNPs may include cationic lipid / cholesterol / DSPC / PEG-DMG at about 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.PREPARATION OF LNPS

[0165] LNPs can be formed, for example, by a rapid precipitation process which entails micromixing the lipid components dissolved in ethanol with an aqueous solution using a confined volume mixing apparatus such as a confined volume T-mixer, a multi-inlet vortex mixer (MIVM), or a microfluidics mixer device as described below. The lipid solution contains one or more cationic lipids, one or more noncationic lipids (e.g., DSPC), PEG-DMG, and optionally cholesterol, at specific molar ratios in ethanol. The aqueous solution consists of a sodium citrate or sodium acetate buffered salt solution with pH in the range of 2-6, preferably 3.5-5.5. The two solutions are heated to a temperature in the range of 25°C-45°C, preferably 30°C-40°C, and then mixed in a confined volume mixer thereby instantly forming the LNP. When a confined volume T-mixer is used, the T-mixer has an internal diameter (ID) range from 0.25 to 1.0 mm. The alcohol and aqueous solutions are delivered to the inlet of the T-mixer using programmable syringe pumps, and with a total flow rate from 10-600 mL / minute. The alcohol and aqueous solutions are combined in the confined-volume mixer with a ratio in the range of 1 : 1 to 1 :3 vol: vol, but targeting 1:1.1 to 1:2.3. The combination of ethanol volume fraction, reagent solution flow rates and t-mixer tubing ID utilized at this mixing stage has the effect of controlling the particle size of the LNPs between 30 and 300 nm. The resulting LNP suspension is twice diluted into higher pH buffers in the range of 6-8 in a sequential, multi-stage in-line mixing process. For26069the first dilution, the LNP suspension is mixed with a buffered solution at a higher pH (pH 6-7.5) with a mixing ratio in the range of 1 : 1 to 1:3 vol: vol, but targeting 1 :2 vol: vol. This buffered solution is at a temperature in the range of 15-40°C, targeting 30-40°C. The resulting LNP suspension is further mixed with a buffered solution at a higher pH, e.g., 6-8 and with a mixing ratio in the range of 1:1 to 1:3 vol: vol, but targeting 1:2 vol: vol. This later buffered solution is at a temperature in the range of 15-40°C, targeting 16-25°C. The mixed LNPs are held from 30 minutes to 2 hours prior to an anion exchange filtration step. The temperature during incubation period is in the range of 15-40°C, targeting 30-40°C. After incubation, the LNP suspension is filtered through a 0.8 pm filter containing an anion exchange separation step. This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL / minute. The LNPs are concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the buffer is exchanged for the final buffer solution such as phosphate buffered saline or a buffer system suitable for cryopreservation (for example containing sucrose, trehalose or combinations thereof). The ultrafiltration process uses a tangential flow filtration format (TFF). This process uses a membrane nominal molecular weight cutoff range from 30-500 KD, targeting 100 KD. The membrane format can be hollow fiber or flat sheet cassette. The TFF processes with the proper molecular weight cutoff retains the LNP in the retentate and the filtrate or permeate contains the alcohol and final buffer wastes. The TFF process is a multiple step process with an initial concentration to a lipid concentration of 20-30 mg / mL. Following concentration, the LNP suspension is diafiltered against the final buffer (for example, phosphate buffered saline (PBS) with pH 7-8, 10 mM Tris, 140 mM NaCl with pH 7-8, or 10 mM Tris, 70 mM NaCl, 5 wt% sucrose, with pH 7-8) for 5-20 volumes to remove the alcohol and perform buffer exchange. The material is then concentrated an additional 1-3 fold via ultrafiltration. The final steps of the LNP manufacturing process are to sterile filter the concentrated LNP solution into a suitable container under aseptic conditions. Sterile filtration is accomplished by passing the LNP solution through a pre-filter (Acropak 500 PES 0.45 / 0.8 pm capsule) and a bioburden reduction filter (Acropak 500 PES 0.2 / 0.8 pm capsule). Following filtration, the vialed LNP product is stored under suitable storage conditions (2°C-8°C, or -20°C if frozen formulation).

[0166] In some embodiments, the LNPs provided herein have a mean geometric diameter that is less than 1000 nm. In some embodiments, the LNPs have mean geometric diameter that is greater 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 between 100-400 nm, 100-300 nm, 100-25026069nm, or 100-200 nm. In some embodiments, the mean geometric diameter is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the mean geometric diameter is between 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the LNPs of a population of LNPs have a diameter that is less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the LNPs of a population of LNPs have a diameter that is 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 of 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 of a population of LNPs have a diameter that is between 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 of a population of LNPs have a diameter that is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm.

[0167] In a particular embodiment, the size of the LNPs ranges between about 1 and 1000 nm, preferably between about 10 and 500 nm, more preferably between about 100 to 300 nm, and preferably 100 nm.THERAPEUTIC AND PROPHYLACTIC COMPOSITIONS

[0168] Provided herein are norovirus vaccine compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, and / or diagnosis of norovirus in humans and other mammals. In some embodiments, the norovirus vaccine compositions comprise a mRNA. In some embodiments, the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 5, for example, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein having a cysteine at amino acid positions 112 and / or 189. In some embodiments, the mRNA comprises SEQ ID NO: 5.

[0169] Norovirus vaccine compositions can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and / or treat infectious disease. In some embodiments, vaccines in accordance with the present disclosure may be used for treatment of norovirus.

[0170] Provided herein are pharmaceutical compositions including norovirus vaccines optionally in combination with one or more pharmaceutically acceptable excipients.26069

[0171] Norovirus vaccines disclosed herein may be formulated or administered alone or in conjunction with one or more other components. For instance, norovirus vaccines (vaccine compositions) may comprise other components including, but not limited to, adjuvants.

[0172] In some embodiments, norovirus vaccines disclosed herein do not include an adjuvant (i. e. , they are adjuvant free). In other embodiments, norovirus vaccines include lipid or polymer-based nanoparticles. In certain embodiments, any of the lipid nanoparticles described herein may act as an adjuvant when combined with norovirus vaccines.

[0173] Aluminum has long been shown to stimulate the immune response against coadministered antigens, primarily by stimulating a TH2 response. It is preferred that the aluminum adjuvant of the compositions provided herein is not in the form of an aluminum precipitate. Aluminum-precipitated vaccines may increase the immune response to a target antigen, but have been show n to be highly heterogeneous preparations and have had inconsistent results (see Lindblad E.B. Immunology and Cell Biology 82: 497-505 (2004)). Aluminum-adsorbed vaccines, in contrast, can be pre-formed in a standardized manner, which is an essential characteristic of vaccine preparations for administration into humans. Moreover, it is thought that physical adsorption of a desired antigen onto the aluminum adjuvant has an important role in adjuvant function, perhaps in part by allowing a slower clearing from the injection site or by allowing a more efficient uptake of antigen by antigen presenting cells.

[0174] The aluminum adjuvant may be in the form of aluminum hydroxide (Al(OH)s), aluminum phosphate (AIPO4), aluminum hydroxy phosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS), or so-called "alum" (KAl(S04)-12H20). See Klein et al., Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)A1 MAS NMR., J. Pharm. Set. 89(3): 311-21 (2000).

[0175] In some embodiments, the aluminum adjuvant is aluminum hydroxyphosphate or AAHS. The ratio of phosphate to aluminum in the aluminum adjuvant can range from 0 to 1.3. In some embodiments, 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 manufactured by blending aluminum chloride and sodium phosphate in a 1 : 1 volumetric ratio to precipitate aluminum hydroxyphosphate. After the blending process, the material is size-reduced with a high-shear mixer to achieve a target aggregate particle size in the range of 2-8 pm. The product is then diafiltered against physiological saline and steam sterilized. See, e g., International Patent Application Publication No. W02013 / 078102.26069

[0176] In some embodiments, the aluminum adjuvant is in the form of amorphous aluminum hydroxyphosphate sulfate AAHS.

[0177] One of skill in the art will be able to determine an optimal dosage of aluminum adjuvant that is both safe and effective at increasing the immune response to the targeted antigenic polypeptides. For a discussion of the safety profile of aluminum, as well as amounts of aluminum included in FDA-licensed vaccines, see Baylor et al., Vaccine 20: S18-S23 (2002). Generally, an effective and safe dose of aluminum adjuvant varies from 150 to 600 pg / dose (300 to 1200 pg / inL concentration). In specific embodiments of the formulations and compositions disclosed herein, there is between 200 and 300 pg aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions disclosed herein, there is between 300 and 500 pg aluminum adjuvant per dose of vaccine.NOROVIRUS VACCINE FORMULATIONS AND METHODS OF USE

[0178] In some embodiments, norovirus vaccines compositions are provided that contain a norovirus vaccine antigen and one or more pharmaceutically acceptable excipients. In some embodiments, the norovirus vaccine compositions are administered to patients. In some embodiments, the norovirus vaccine compositions comprise any norovirus vaccine antigen disclosed herein (e.g., an mRNA sequence or a VLP). In some embodiments, the norovirus vaccine that is part of the norovirus vaccine composition comprises an mRNA. In some embodiments, the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 5, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein having a cysteine at amino acid positions 112 and / or 189. In some embodiments, the norovirus vaccine that is part of the norovirus vaccine composition comprises SEQ ID NO: 5.

[0179] In some embodiments, combinations are provided that comprise norovirus vaccine compositions and at least one additional active substance, such as, for example, a prophylactically-active substance, or a combination of both. The combinations may be sterile, pyrogen-free, or both sterile and pyrogen-free.

[0180] General considerations in the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.

[0181] In an aspect, formulations of norovirus vaccine compositions are disclosed herein. In some embodiments, the formulations are prepared by any method known or hereafter developed26069in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., one or more polynucleotides (mRNA)) into association with an excipient and / or one or more other accessory' ingredients, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the product into a desired single- or multi -dose unit. In some embodiments, the active ingredient in the formulations disclosed herein comprises any mRNA disclosed herein. In some embodiments, the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 5, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein having a cysteine at amino acid positions 112 and / or 189. In some embodiments, the norovirus vaccine that is part of the norovirus vaccine composition comprises SEQ ID NO: 5.

[0182] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in compositions, combinations, and formulations described herein will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition, combination, or formulation may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%. between 5-80%, or at least 80% (w / w) active ingredient. In some embodiments, the active ingredient comprises any mRNA described herein. In some embodiments, the active ingredient comprises an mRNA that is at least 90% identical to SEQ ID NO: 5, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein having a cysteine at amino acid positions 112 and / or 189. In some embodiments, the active ingredient comprises SEQ ID NO: 5.

[0183] Some aspects of the present disclosure provide formulations of the norovirus vaccine, wherein the vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a norovirus antigenic polypeptide). An “effective amount” of a norovirus RNA vaccine is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g.. size, and extent of modified nucleosides) and other components of the norovirus RNA vaccine, and other determinants. In general, an effective amount of the norovirus RNA vaccine composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a26069corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen.Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

[0184] In some embodiments, the antigen-specific immune response is characterized by measuring an anti -norovirus antigenic polypeptide antibody titer produced in a subject administered a norovirus vaccine as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., a norovirus antigenic polypeptide, such as a VP1 or VP2 polypeptide) or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

[0185] In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by the norovirus vaccine.MODES OF VACCINE ADMINISTRATION

[0186] Norovirus vaccines described herein may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the norovirus vaccines that are administered comprise an mRNA sequence. In some embodiments, the mRNA sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein having a cy steine at amino acid positions 112 and / or 189. In some embodiments, the mRNA sequence comprises SEQ ID NO: 5

[0187] A prophylactically effective dose is a therapeutically effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments the therapeutically effective dose is a dose listed in a package insert for the vaccine. In some embodiments, the26069amount of vaccine of the present disclosure provided to a cell, a tissue, or a subject may be an amount effective for immune prophylaxis.

[0188] Norovirus vaccines may be formulated for administration by any route which results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, intratracheal, and / or subcutaneous administration. In some embodiments, norovirus vaccines are administered intramuscularly.

[0189] The present disclosure provides methods comprising administering vaccines 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, and the like.

[0190] Norovirus vaccine compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of vaccine compositions may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0191] Norovirus vaccines may be administrated with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster’ refers to an extra administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours. 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours. 11 hours. 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 826069months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In some embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0192] Norovirus vaccines may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the norovirus vaccines may be utilized to treat and / or prevent a variety of noroviruses. Vaccines have superior properties in that they produce much larger antibody titers and produce responses earlier than commercially available anti-viral agents and compositions.METHODS OF TREATMENT

[0193] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for prevention and / or treatment of norovirus in humans and other mammals.

[0194] In an aspect, a method of treating or preventing a disorder related to norovirus infection is provided. In some embodiments, the method comprises administering to a subject in need any of the polypeptides or VP1 proteins described herein.

[0195] In some embodiments, the method comprises administering to a subject a polypeptide that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions 112 and 189 of SEQ ID NOs: 1, 7, and 11 is a cysteine. In some embodiments, the polypeptide comprises SEQ ID NO: 1.

[0196] In some embodiments, the method comprises administering to a subject in need any of the polynucleotides described herein. In some embodiments, the polynucleotide comprises a sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 3, 5. 9, and 13, wherein the polynucleotide encodes a VP1 protein comprising any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs: 3, 5, 9, and 13.

[0197] In some embodiments, the method comprises administering to a subject in need any of the compositions described herein. In some embodiments, the compositions comprise any of the polypeptides or any of the polynucleotides described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a polypeptide sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein one or both of the amino acid residues at positions 112 and 189 of SEQ ID NOs: 1, 7, and 11 is a cysteine. In some26069embodiments, the polypeptide comprises any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the composition comprises a polynucleotide that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 3, 5, 9, and 13, wherein the polynucleotide encodes a VP1 protein comprising any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the composition comprises any one of SEQ ID NOs: 3, 5, 9, and 13.

[0198] In some embodiments, the method comprises administering to a subject in need any of the vectors disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 3, 9, and 13, wherein the polynucleotide encodes a VP1 protein comprising any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the vector comprises any one of SEQ ID NOs: 3, 9, and 13.

[0199] In some embodiments, the method comprises administering to a subject in need any of the norovirus vaccines described herein. In some embodiments, the norovirus vaccine comprises an mRNA polynucleotide described herein. In some embodiments, the mRNA polynucleotide comprises a sequence that is at least 90% or at least 95% identical to SEQ ID NO: 5, wherein the mRNA encodes a VP1 protein comprising any one of SEQ ID NOs: 1, 7, and 11. In some embodiments, the mRNA polynucleotide comprises SEQ ID NO: 5.

[0200] In some embodiments, norovirus vaccines disclosed herein are used for prevention and / or treatment of norovirus in human and other animals. In some embodiments, the norovirus vaccines comprise an mRNA sequence.

[0201] Norovirus vaccines disclosed herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and / or treat infectious disease. In exemplary aspects, the norovirus vaccines of the present disclosure are used to provide prophylactic protection from norovirus. Prophylactic protection from norovirus can be achieved following administration of a norovirus vaccine of the present disclosure. Vaccines can be administered once, twice, three times, four times, or more. It is possible, to administer the vaccine to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

[0202] In some embodiments, the norovirus vaccines of the present disclosure are useful in a method of preventing a norovirus infection in a subject, the method comprising administering to said subject at least one norovirus vaccine as provided herein. In some embodiments, the norovirus vaccines of the present disclosure are useful in a method of inhibiting a primary norovirus infection in a subject, the method comprising administering to said subject at least one norovirus vaccine as provided herein. In some embodiments, the norovirus vaccines of the present disclosure are useful in a method of treating a norovirus infection in a subject, the method26069comprising administering to said subject at least one norovirus vaccine as provided herein. In some embodiments, the norovirus vaccines of the present disclosure are useful in a method of reducing an incidence of norovirus infection in a subject, the method comprising administering to said subject at least one norovirus vaccine as provided herein. In some embodiments, the norovirus vaccines of the present disclosure are useful in a method of inhibiting spread of norovirus from a first subj ect infected with norovirus to a second subj ect not infected with norovirus, the method comprising administering to at least one of said first subject and said second subject at least one norovirus vaccine as provided herein.

[0203] In some embodiments, a method of eliciting an immune response in a subject against a norovirus is provided. The method involves administering to the subject a norovirus vaccine described herein, thereby inducing in the subject an immune response specific to norovirus antigenic polypeptide or an immunogenic fragment thereof.EXAMPLES

[0204] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Example 1. Identifying Residues to Mutate to Stabilize VLPs

[0205] The goal of this work was to engineer disulfide bonds that could stabilize the T=3 icosahedral structure of virus like particles (VLPs) from norovirus GII genoty pes. Through sequence alignment and modeling across the strains of GII.2, GII.3, GII.4, and GII.6, residues 112 and 189 of the norovirus VP1 protein were identified as potential residues that could be mutated. The mutations N112C and G189C were introduced into several different genotypes of the GII genogroup of norovirus (“DS1 mutants’', see Example 8) in attempt to stabilize the resulting VLPs following recombinant expression of VP1.Example 2. Expression and Analysis of VP1 Proteins

[0206] Plasmids encoding either WT or DS variant VP1 proteins w ere transiently transfected into Expi 293F cells (ThermoFisher) using Expifectamine (ThermoFisher) following manufacturer's recommended protocol. Bulk cell cultures were harvested 72 hours posttransfection and frozen at -70°C. To generate clarified bulk supernatant, the bulk harvests were thawed at room temperature, mixed thoroughly by inversion, and centrifuged at 3500 xg for 1026069minutes at room temperature. The resulting clarified bulk supernatant was transferred to a clean, sterile tube and stored at -70°C until further processing and characterization.

[0207] 15 pg of clarified cell culture lysate and 2.6 pg of clarified cell culture supernatant, as determined by Bradford assay, were resolved on a 4-12% gradient, 1.5 mm, 15-well Bis-Tris NuPAGE gel in IX MES buffer (Invitrogen, ThermoFisher). SeeBlue Plus2 pre-stained protein standard (ThermoFisher) was included on the SDS-PAGE for molecular weight reference.Proteins were transferred onto nitrocellulose membranes using the iBlot western blotting system (Invitrogen, ThermoFisher). Membranes were transferred to the Bandmate Automated Western Blot Processor (Invitrogen) for blocking and probing. Membranes were blocked for 1 hour at room temperature with 30 mL of 5% non-fat dry milk (Blotting-grade blocker, Bio-Rad) in IX tris-buffered saline containing 0.1% Tween-20 (IX TBST). Membranes were probed with a primary antibody cocktail of 2 mouse norovirus VP1 monoclonal antibodies at a dilution of 1 : 1000 in 5% non-fat dry’ milk in IX TBST for 1 hour at room temperature. The primary antibody cocktail consisted of a 1: 1 mixture of two mouse norovirus VP1 monoclonal antibodies: My’ BioSource.com clone # M120539 (cat # MBS832466) and Abeam clone # NVGC-01 (cat # ab272687). Membranes were probed with an alkaline-phosphatase-conjugated affinipure goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, cat # 1115-055-146) diluted 1: 1000 in 5% non-fat dry’ milk in IX TBST for 1 hour at room temperature. Membranes were washed 3 times for 5 minutes each at room temperature with IX TBST. Membranes were developed using 1-step NBT / BCIP (ThermoFisher) according to manufacturer's recommendations.

[0208] VP1 protein for GII.4-WT and GII.4-DS1 was readily visible in the supernatant by Coomassie-stained SDS-PAGE gel and anti-VPl western blot whereas VP1 protein for GII.3 and GII.6 WT and DS1 variants in the supernatant was primarily visible by anti-VPl western blot only (Figure 1 A). VP1 protein is visible in the cell lysate by both Coomassie-stained SDS-PAGE gel and anti-VPl western blot for all Norovirus strains (Figure IB). Protease cleavage fragments are present for wildtype and DS1 variants for GII.3 and GII.6 in both the supernatant and cell lysate fractions (Figure 1A and IB).

[0209] To evaluate the effect of the DS1 mutation on norovirus VLP, we moved forw ard w ith strains GII.3 and GII.6. Clarified bulk lysates containing VP1 wild type and VP1 mutant proteins GII.3 DS1 (SEQ ID NO: 1), GII.3 WT (SEQ ID NO: 2), GII.6 DS1 (SEQ ID NO: 7), GII6. WT (SEQ ID NO: 8) were carried forw ard into Example 3 below.26069Example 3. Sucrose Gradient and Diamide Experiments

[0210] Bulk harvested and clarified Expi293 cell lysates were then concentrated and analyzed via sucrose gradient ultracentrifugation to determine if the expressed VP1 protein form VLPs.

[0211] Norovirus VLPs typically elute near the center of the 15-36% gradient (-25% sucrose). We observed VLP formation from expressed VP1 protein of genotypes G11.3 and G1I.6 comprising the DS1 mutations (GII.3 DS1 and GIL 6 DS1). Notably, compared to wild-type, the GIL 3 DS1 mutant showed much stronger bands in VLP fractions, possibly due to disulfide-induced VLP stabilization. FIG. 2A shows sucrose gradient purification of GII.3 and GII.6 VLPs with and without DS1 mutations. Fractions 5, 6. and 7 for these genotypes were pooled and dialyzed to remove sucrose and concentrated to 0.5 mg / mL prior to all subsequent analyses.

[0212] To determine if disulfide bonds were formed in the DS1 mutants, GII.3 and GII.6 VLPs were run on SDS-PAGE under three conditions: 1) without additional treatment, 2) after treatment with 20 mM beta-mercaptoethanol (reducing) for 1 hour, and 3) after treatment with 20 mM diamide (oxidizing) for 1 hour (FIG. 2C).

[0213] Overall, the data show a distinct shift was observed in the VP1 bands from the DS1 mutants from GII.3 and GII.6 under oxidizing conditions, which was largely not observed for the wild-type VP1 proteins from GII.3 and GII.6, indicating formation of the engineered disulfide bonds (see FIG. 2C, right panel). Bond formation was only observed on SDS-PAGE after treatment with a strong oxidizing agent; however, there may be a minor amount of VP1 material in the well in the untreated condition. All subsequent experiments were performed without diamide pretreatment (i.e., in the untreated condition). Sucrose-gradient purified VLPs from Example 3 were carried forward in to Examples 4 and 5 below.Example 4. Dynamic Light Scattering (PLS) and NanoDSF

[0214] To measure the effects of the DS1 mutation on VLP stability, dynamic light scattering (DLS) and differential scanning fluorimetry (DSF) experiments were performed. Compared to wild-type, GII.3 DS1 mutation led to reductions in both the hydrodynamic diameter (67.8 to 58.6 nm) and poly dispersity' index (0.18 to 0.10), indicating GII.3 DS1 VLPs are, on average, more compact and more homogenous compared to wild-type GII.3 VLPs (FIG. 2B). The DS1 mutation did not appear to have a significant effect in the GII.6 background as measured by DLS.Compared to wild-type, GII.6 DS1 only led to slight reductions in the hydrodynamic diameter (61.6 to 59.2 nm) and a marginal increase in the poly dispersity index (0.14 to 0.16) (FIG. 2B). We also conducted nanoDSF experiments to measure the thermal stability of VLPs. Wild-type GII.3 and GII.6 VLPs both showed monophasic transitions with melting temperatures (Tm) of64.0 and 66.8 °C, respectively (FIGs. 2D and 2E). FIG. 2D shows nanoDSF measurements of GII.3 and GII.6 VLPs. Each line represents an average of three technical replicates. FIG. 2E provides quantification of melting temperature (Tm) from nanoDSF experiments, showing a significant increase in thermal stability- of GII.3 VLPs upon introduction of the DS1 mutation.

[0215] For GII.3, the DS 1 mutation increased the Tm by 4.2 °C, however for GII.6, the DS 1 mutation only increased the Tm by 1.0 °C. Finally, compared to the wild-type VLPs, we note that the DS1 mutation led to an ~2-fold increase in final VLP yield for GII.3 and no significant increase in yield for GII.6 (FIG. 2F).

[0216] Together, these data indicate that the DS1 mutation significantly stabilizes GII.3 VLPs. whereas no significant stabilization was observed for GII.6 VLPs.Example 5. Stability of the VLPs / Negative Stain Electron Microscopy(neSM)

[0217] To further assess VLP formation and stability, purified VLPs were imaged via negativestain electron microscopy (nsEM). Sucrose gradient purified VLPs from genotypes GII.3 WT, GII.3 DS1, GII.6 WT and GII.6 DS1 were directly compared using nsEM after a single freezethaw ((FIG. 3A (GII.3 WT genoty pe); FIG. 3B (GII.3 DS1 genoty pe); FIG. 3C (GII.6 genoty pe); FIG. 3D (GII.6 DS1 genotype)) or without freezing (FIG. 4). The stabilizing effect was less pronounced when comparing VLPs without a freeze-thaw cycle, which showed fewer breakdown products in the WT GII.3 sample (FIG. 4).

[0218] Overall, FIGs. 3A-3D show that the DS1 mutation leads to more uniform GII.2 and GII.6 VLPs. Intact GII.3 and GII.6 VLPs appeared to be consistent with T=3 icosohedral symmetry.Example 6. Binding Assays

[0219] To assess the ability- of GII.3WT and GII.3DS1 VLPs to recognize / bind to histo-blood group glycans present in human saliva, we performed a binding affinity assay in which we tested the binding affinity of freshly prepared WT VLPs and DS1 VLPs to a panel of human saliva samples (n=51). Also, a panel of GII.3-specific monoclonal antibodies (mAbs, n=20) was screened for their ability- to bind GII.3 and GII.3-DS1 VLPs.

[0220] Binding activities of GII.3 DS1 or GII.3 WT VLPs to human saliva samples were analyzed. ELISA plates were coated with 51 human saliva samples, and then incubated with 20 pg / mL GII.3 WT or DS1 VLPs. Bound VLPs were then detected by polyclonal sera against GII.3. Relative luminescent units of WT and DS1 are reported on the X axis and Y axis, respectively (FIGs. 5A and 5B). Data were fitted into a simple linear regression model usingGraphPad Prism software. Data shown in the box (dashed line) in FIG. 5 A are shown in a different scale in FIG. 5B.

[0221] Binding activities of GII.3 DS1 or GII.3 WT VLPs to monoclonal antibodies were analyzed. ELISA plates were coated with 50 ng of either GII.3 or GII.3-DS1 VLP and incubated with 20 unique human monoclonal antibodies starting at 4 pg / ml and diluted four-fold, ten-point dilution series. Bound antibodies were detected using goat anti -human Ig - Fc fragment conjugated to horseradish peroxidase (HRP). Two (2) antibodies showed no binding to either GII.3 or GII.3 DSL Interpolated endpoint titer for each antibody against either GII.3 or GII.3 DS1 are reported on the X and Y axes, respectively. Data are fitted into a simple linear regression model using GraphPad Prism software.

[0222] Results indicate that both GII.3 WT VLPs and GII.3DS1 VLPs demonstrate similar binding properties to saliva samples (FIGs. 5A and 5B) (R2= 0.9208). However, there is a clear difference between the ability of GII.3 WT and GII.3DS1 VLPs to bind to human saliva when comparing freshly prepared room temperature VLPs to VLPs stored overnight @ 4°C. The binding ability of GII.3DS1 w as not hindered in minimal buffer (PBStw) under both storage conditions, whereas the ability' of GII.3 WT VLPs to efficiently bind human saliva was dramatically decreased with overnight storage at 4°C (FIGs. 6A and 6B). In addition, these results are further supported by DLS stability analysis showing the breakdown of GII.3 WT VLPs over extended storage at 4°C.

[0223] Results show' that GII.3 and GII.3-DS1 VLPs bind to the same human saliva samples and mAbs with similar avidities (R2= 0.9208 and 0.8031, respectively) (FIGs. 5A and 5B, and FIG. 8).Example 7. LNP Encapsulation of mRNAs

[0224] LNPs encapsulating the GII.3 DS1 mRNA (SEQ ID NO: 5) and GII.3 WT mRNA (SEQ ID NO:6) were formed via rapid precipitation using microfluidics or tee-mixers to micro-mix two fluid streams. One fluid stream contained lipids dissolved in ethanol and the other fluid stream was an aqueous solution containing mRNA. The lipid solution prepared in ethanol contained cationic lipid, cholesterol, PEG-DMG, and phospholipid (DSPC) at a specified molar ratio. The aqueous solution consisted of a 10 mM sodium citrate buffer, pH 4.5-5.5 and contained mRNA.

[0225] For microfluidic mixed LNPs, the mRNA and lipid containing solution streams were combined at 12 mL / min in a 3: 1 ratio to produce a 25 vol: vol% alcohol in the mixed solution. The resulting LNP suspension had an N / P ratio in the range of 5-8. The mRNA / LNP suspension underwent buffer exchange and ethanol removal via dialysis. mRNA / LNP suspensions w eredialyzed 3x against 20 mM Tris, 10% sucrose (w / v), pH 7.5 at volumes of 100-200x of the product using SpectraPor® Float- A-Lyzers® (Repligen) with a molecular weight cutoff of 100 kDa. The dialyzed mRNA / LNP suspension was then filtered through 0.2 pm polyethersulfone (PES; Pall) or poly vinylidene fluoride (PVDF; Millipore) sterile filters into sterile glass vials and sealed. The mRNA / LNP formulation was then stored under refrigerated (2-8°C) or frozen (20°C or <-60°C) conditions. Prior to use, the mRNA / LNP vials were equilibrated to room temperature.

[0226] For tee-mixer prepared mRNA / LNPs, the lipid containing solution stream was heated to a temperature in the range of 35-40°C and then was mixed with the mRNA containing solution stream in a confined volume mixer (ID 0.5 mm) using programmable pumps. The total flow rate at the inlet of the t-mixer ranged from 100-150 mL / min and delivered the streams in a ratio of 0.90: 1 to 1.3:1 to produce 43-53 vol-vol% alcohol in the mixed solution. The resulting mRNA / LNP suspension had an N / P ratio in the range of 5-8 and was diluted 2x into higher pH buffers (pH range 6-8) utilizing a sequential, 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 mixing ratio of 1 : 1 vol: vol. The buffer solution was held at a temperature in the range of 35-40°C. The mRNA / LNP formulation was then further mixed with mDPBS, pH of 7.5 at a mixing ratio of 1: 1 vol: vol. The mDPBS, pH 7.5 solution was at a temperature in the range of 16-25°C. The resulting mRNA / LNP suspension was held for 30 min in a temperature range of 16-25°C prior to undergoing anionic exchange filtration. After filtration, the mRNA / LNP suspension was then concentrated and diafiltered via an ultrafiltration process to remove alcohol and perform a buffer exchange into the final buffer solution. 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. Ultrafiltration was first used to concentrate the mRNA / LNP suspension 6-8-fold by volume, targeting a mRNA concentration of 0.4-0.7 mg / mL. Alcohol was removed through subsequent diafiltration (10 diavolumes) using 20 mM Tris. 10% sucrose (w / v), pH 7.5. The mRNA / LNP suspension was then further concentrated 3-fold 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 pm and 0.2 pm PES, CA and / or PVDF filters. The bioburden reduced monovalent mRNA / LNP suspension is then stored under frozen (<-60°C) conditions until further use. Prior to terminal 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 pm and 0.2 pm PES and / or PVDF filters. After filtration and under aseptic conditions, the mRNA / LNP suspension was diluted to a final target mRNA concentration26069utilizing 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 frozen conditions (<-60°C). Prior to administration, the mRNA / LNP vials were equilibrated to room temperature.Example 8. mRNA Studies - Antibodies elicited by GII.3-DS1 mRNA immunization block HGBA glycan binding better than those elicited by GII.3 immunization

[0227] To investigate whether DS1 also elicits higher immune responses than WT as an mRNA vaccine antigen, we synthesized mRNAs encoding GII.3 DS1 (SEQ ID NO: 5) and GII.3 WT (SEQ ID NO: 6) and tested mRNAs encapsulated in LNP (as described in Example 7) in mice (FIGs. 7A-7C) (p values shown in Tables 1 and 2) FIGs. 9A-9B) (p values shown in Table 3. Mice were immunized with three dose groups that included the LNPs with encapsulated SEQ ID NO: 5 and LNPs with encapsulated SEQ ID NO: 6) (used as a control), twice at four-week intervals. Serum was collected at 2 weeks post doses 1 and 2, and evaluated for antibody binding and HBGA blockage titers (FIG. 7A). Antibody titers were measured by ELISA binding and histo-blood group antigen (HBGA) blockage, 2 w eeks after each immunization.

[0228] Briefly, BALB / c mice (N = 10 per group) were immunized with 0.1 pg, 0.5 pg or 2 pg mRNAs at weeks 0 and 4. Blood draws were performed at weeks 2 and 6 (FIG. 7A).

[0229] FIGs.7B and 7C: Sera from mice immunized with mRNAs encoding GII.3 DS1 or GII.3 WT were assayed for the presence of GII.3 DS1 binding antibodies (FIG. 7B) or HBGA blockade antibodies (FIG. 7C). Limits of detection are indicated with horizontal dotted lines (titer = 50 in B and 20 in C). Geometric mean titers with geometric standard deviation are shown in scatter dot plot. P values were determined by two-tailed unpaired t-test using GraphPad Prism software (p < 0.05 = *, p < 0.01 = **).

[0230] FIGs.9A and 9B: Week 6 sera were assayed for the presence of GII.3 binding antibodies (FIG. 9A) or HBGA blockade antibodies (FIG. 9B). Limits of detection are indicated with horizontal dotted lines (titer = 50 in FIG. 9A and 20 in FIG. 9B). Empty lipid nanoparticle (LNP) was immunized as a negative control. Geometric mean titers with geometric standard deviation are shown in scatter dot plot. P values w ere determined by tw o-tailed unpaired t-test using GraphPad Prism software (p < 0.01 = **).

[0231] The data show that mRNAs encoding GII.3 DS1 (SEQ ID NO: 5) at low (0.1 pg) and medium (0.5 pg) dose groups elicited higher VLP binding titers than mRNAs encoding GII.3 WT (SEQ ID NO: 6) at both week 2 and w eek 6 (FIG. 7B). The difference was not observed at the high (2 pg) dose group (FIG. 7B). At week 2, the low7dose of either DS1 or WT mRNAs did not elicit strong enough antibody titers measurable by the HBGA blockage assay (FIG. 7C). Both the26069median and high dose groups showed significant differences with the mRNAs encoding DS1 inducing higher HBGA blockage titers (FIG. 7C). At week 6, 0.1 pg DS1 mRNAs elicited higher blockage titers than WT while at 0.5 or 2 pg DS1 and WT mRNAs performed similarly (FIG. 7C). Week 6 data remained consistent whether the assay reagent VLP used GII.3 or GII-DS1 (FIGs. 9A and 9B) Taken together, these data indicate that mRNA encoding GII.3 DS1 is more immunogenic than mRNA encoding GII.3 WT at non-saturating dose levels in mice.

[0232] Tables 1, 2, and 3 show the P values from the data reported in FIGs. 7B and 7C and FIGs. 9A and 9B. P values were determined by two-tailed unpaired t-test using GraphPad Prism software. In FIGs. 7B, 7C. 9A, and 9B, P values of < 0.05 are denoted as * and P values of < 0.01 are denoted as **.Table 1. P Values from FIG.7BTable 2. P Values from FIG.7CTable 3. P Values from FIGs.9A and 9B26069Example 9. Materials and MethodsPlasmid Construction and VLP Expression

[0233] Nucleotide sequences for mammalian expression of norovirus VP1 capsid from genotypes GII.3 (Genbank Accession MT409884), GII.6 (Genbank Accession MH260487) and Gil.4 (G1I.4: Genbank Accession MK754446) were mammalian codon-optimized and subcloned into a eukaryotic-expression vector under the control of the CMV promotor. DS1 variants were generated by introducing cysteine substitutions at amino acid positions N112 and N189 within the VP1 protein of each genotype. Plasmids were transiently transfected into Expi 293F cells (ThermoFisher) using Expifectamine (ThermoFisher) following the manufacturer’s recommended protocol. Bulk cell cultures were harvested 72 hours post-transfection and frozen at -70°C. To generate clarified bulk supernatant, the bulk harvests were thawed at room temperature, mixed thoroughly by inversion, and centrifuged at 3500 xg for 10 minutes at room temperature. The resulting clarified bulk supernatant was transferred to a clean, sterile tube and stored at -70°C.VLP Purification

[0234] The frozen cell culture was thawed and clarified through a Sartopure GF+ (Sartorius) depth filter. The clarified VLPs were precipitated using 1.5 M ammonium sulfate (AS) and pelleted by centrifugation at 5000 xg for 10 min. The pellet was resuspended in an anion exchange chromatography (AEX) loading buffer and filtered through a 0.2 pm PVDF membrane (Millipore). The filtered AS precipitation product was purified by AEX using a Sartobind Q membrane (Sartorius) and the VLPs were eluted in an elevated ionic strength buffer. The AEX eluates was further purified by size exclusion chromatography (SEC) using a Sepharose 6 FF (Cytiva) column. Selected SEC fractions were pooled, concentrated, and exchanged into a formulation buffer using a 300 kDa UF / DF membrane (Repligen). The UF / DF product was spiked with a cryoprotectant and filtered through a 0.2 pm PVDF membrane (Millipore). All purified VLPs were frozen and stored at -70° C. The final concentration of the VLPs was about 1 mg / rnL as measured by the Bradford Assay (ThermoFisher) using the manufacturer’s recommended protocol.SDS-PAGE and Western Blot

[0235] 15 pg of clarified cell culture lysate and 2.6 pg of clarified cell culture supernatant, as determined by Bradford assay, were resolved on a 4-12% gradient, 1.5 mm, 15-well Bis-Tris NuPAGE gel in IX MES buffer (Invitrogen, ThermoFisher). SeeBlue Plus2 pre-stained protein26069standard (ThermoFisher) was included on the SDS-PAGE for molecular weight reference.Proteins were transferred onto nitrocellulose membranes using the iBlot western blotting system (Invitrogen, ThermoFisher). Membranes were transferred to the Bandmate Automated Western Blot Processor (Invitrogen) for blocking and probing. Membranes were blocked for 1 hour at room temperature with 30 mL of 5% non-fat dry milk (Blotting-grade blocker, Bio-Rad) in IX tris-buffered saline containing 0.1% Tween-20 (IX TBST). Membranes were probed with a primary antibody cocktail of 2 mouse norovirus VP1 monoclonal antibodies at a dilution of 1 : 1000 in 5% non-fat dry milk in IX TBST for 1 hour at room temperature. The primary antibody cocktail consisted of a 1: 1 mixture of two mouse norovirus VP1 monoclonal antibodies: My BioSource.com clone # M120539 (cat # MBS832466) and Abeam clone # NVGC-01 (cat # ab272687). Membranes were probed with an alkaline-phosphatase-conjugated affinipure goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, cat # 1115-055-146) diluted 1 : 1000 in 5% non-fat dry’ milk in IX TBST for 1 hour at room temperature. Membranes were washed 3 times for 5 minutes each at room temperature with IX TBST. Membranes were developed using 1-step NBT / BCIP (ThermoFisher) according to manufacturer’s recommendations.Sucrose Gradient Purification

[0236] 30 mL frozen bulk lysates were thawed in a water bath, clarified by centrifugation, and concentrated to 1 mL using an Amicon 100 kDa MWCO centrifugal filter unit. 5 pL of input material was saved for SDS-PAGE. 16mL 15-36% continuous sucrose gradients were made on the day of purification using a Hopfner SG15 gradient maker connected to a Cole Palmer Master Flex L / S Digital peristaltic pump flowed at 3.6 mL / min using size 16 tubing capped with a blunt 4-inch 14-gauge needle placed at the bottom of a 17mL ultracentrifuge tube. 1 mL of concentrated bulk lysate was gently layered on top and gradients were placed in SW28.1 buckets and loaded into a SW28Ti swinging bucket rotor. Samples were centrifuged for 3 hours at 28,000 rpm in a Beckman Optima CL-100K Ultracentrifuge operated at 4 °C under vacuum. Gradients were manually fractionated from the top down using a pipette into 12 x 1.34 mL fractions. 15 pL of each fraction was analyzed on a 4-12% SDS-PAGE gel and norovirus VLPs consistently eluted in the center of the gradient. Fractions 5-7 were pooled and dialyzed overnight using a 25 kDa MWCO dialysis cassette against norovirus storage buffer containing: 25 mM Tris pH 7.5.150 mM NaCl, 0.02% PS-80, and 5% sucrose. Protein concentration was estimated by Bradford assay using a BSA standard curve, and concentrations were adjusted to ~0.5 mg / mL for use in subsequent assays. Proteins were aliquoted, flash frozen in liquid ISLand stored at -80 °C.26069Dynamic Light Scattering (DLS)

[0237] The hydrodynamic diameter and polydispersity index (Pdl) of purified VLPs was measured using a Malvern Zetasizer Nano ZS instrument. Measurements were recorded using 0.1 mL of purified VLPs at 0.5 mg / mLin norovirus storage buffer in a ZEN040 cuvette using the following instrument parameters:- Material = Protein (RI=1.450, Absorbtion=0.001)- Dispersant = ICN PBS Tablets (Viscosity=0.8882cP, RI=1.330)- Temperature = 25 °C- Method = Mark-Houwink- Cell type = ZEN0040- Equilibration time = 60 seconds- Back scatter = 173°- 3 measurements each (automatic duration)

[0238] General purpose data analysis was performed in Zetasizer software.Nano Differential Scanning Fluorimetry (nanoDSF)

[0239] Thermal stability assays were performed using a NanoTemper Prometheus NT.48 nanoDSF instrument. Purified VLPs at a concentration of 0.5 mg / mL in norovirus storage buffer were loaded into 3 NanoTemper high-sensitivity capillaries (3 technical replicates each). An initial discovery scan was used to optimize excitation power at 280 nm. Temperature was ramped from 20 °C to 95 °C at a rate of 1 °C per minute and fluorescence was recorded at 330 nm and 350 nm. The 330 / 350 fluorescence ratio and its derivative were used to fit the inflection point (Tm). Average 330 / 350 fluorescence signal of the 3 replicates was plotted in GraphPad Prism software for visualization.Electron Microscopy

[0240] Purified VLP samples along with norovirus storage buffer were shipped to Nano Imaging Services (NIS, San Diego, CA) for negative stain electron microscopy analysis. The samples were diluted to a final concentration of 0.012 mg / mL with buffer. Samples were deposited on a layer of continuous carbon supported by nitro-cellulose on a 400-mesh copper grid. The grids were prepared by applying 3 pL of sample suspension to a cleaned grid, blotting away with filter paper, and immediately staining with uranyl formate. Samples were imaged on an FEI Tecnai T12 electron microscope (serial number DI 100), operating at 120keV and equipped with an FEI Eagle 4k x 4k CCD camera. Negative stain grids were transferred into the26069electron microscope using a room temperature stage. Data-collection was carried out using Leginon software (Suloway et al., 2005 and Cheng et al., 2021), where high magnification images are acquired by selecting targets at a lower magnification. Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen. After identifying potentially suitable target areas for imaging at lower magnifications, high magnification images were acquired at nominal magnifications of 110,000x (0.099 nm / pixel), 52,000x (0.212 nm / pixel) and 21,000x (0.513 nm / pixel). The images w ere acquired at a nominal underfocus of -5.0 gm to -1.2 pm and electron doses of -10-25 e- / A2. Representative micrographs for each sample collected at 52.000x were selected for qualitative comparisons. All micrographs from the 52,000x magnification w ere imported into RELION (v4.0.1) and CTF corrected using CTFFIND (v4.1.14). VLPs were manually picked from micrographs and were extracted in a 300 x 300 pixel (636 x 636 A) box and subjected to 2D classification using 4 classes and a particle diameter of 500 A.Salvia Binding Affinity Assay

[0241] 384 well Maxisorp assay plates (Thermo Fisher, Scientific) were coated with 25 pL of human saliva (Precision for Medicine) diluted 1:500 in DPBS. Plates were sealed with aluminum foil and centrifuged @ 1500 x g for 1 minute and incubated overnight at 4°C in a humidity chamber (21 °C, 80% RH). Norovirus VLPs were diluted in 5% Nonfat Powder Dry Milk (NFPDM) in PBS with 0.1% tween20 (PBS-T) to a starting concentration of 20 pg / mL and then diluted in a two-fold, ten-point serial dilution. The masterblock was sealed with aluminum foil and centrifuged (a 2000 rpm for a minute. The plates w ere stored overnight at 4°C in a humidify chamber (21°C, 80% RH). The assay plates were rinsed 6X with IX PBS-T (100pl / well) with blue washer dispenser (Blue Cat Bio). The assay plates with human saliva samples were blocked with 5% NFPDM PBS-T (80pl) dispensed with a multidrop combi (Thermo Fisher Scientific) and incubated for 30 min in a humidified incubator (21 °C, 80% RH). VLPs were added to 384 well assay plates, incubated for Ih in a humidified incubator to allow the VLPs to bind to the human saliva (21 °C, 80% RH). After incubation, the assay plates were washed (6X PBS-T, lOOpl / well). Saliva-bound VLP w as detected with 20pl of GIL 3 specific polyclonal rabbit serum in 5% NFPDM PBS-T(l:3000) for Ih in a humidified incubator (21°C, 80% RH). Assay plates were rinsed 6X with PBS-T. (lOOpl / well) followed with the addition of 20pl / well of Goat anti rabbit-HRP IgG (1:5000 in 5% NFPDM PBS-T) (Jackson Immunoresearch). Assay plates were incubated for Ih in a humidified incubator (21°C, 80% RH). After incubation, the assay plate was washed 6X PBST, (lOOpl / well) and luminescence w as developed with 20pl / well of Luminescentsubstrate (West Pico PLUS, Thermo Scientific, Pierce) for 15 min at RT. Ultrasensitive luminescence was read on an Envision plate reader at 0.1 seconds per well.Serum IgG ELISA Assay

[0242] Maxisorp 384-well plates were coated with 50 ng / well of VLPs. Assay plates were centrifuged @ 1500 xg for 1 minute and incubated overnight at 4°C in a humidity chamber (21°C, 80% RH). Assay plates were washed 6X with PBS-T and blocked with 3% NFDM PBS-T for 30 minutes, followed by additional washes 6X with PBS-T. Serum samples diluted ten times in 4-fold serial dilutions in 3% NFPDM PBS-T were added to assay plates and incubated for 2 hours in a humidified incubator (21°C, 80% RH). Goat anti-mouse IgG (Fc)-HRP (Jackson ImmunoResearch) was added to assay plates and incubated for 1 hour in a humidified incubator (21 °C, 80% RH). Plates were washed with 6X with PBS-T and luminescent signal developed with West Pico PLUS Chemiluminescent Substrate and read on an EnVision plate reader (PerkinElmer, Waltham, Massachusetts, USA). Interpolated serum antibody endpoint titers were calculated as the highest dilution where RLU signal is above a 50000 RLU cutoff. Samples where no dilution crossed the threshold were given a placeholder titer of "25”. For samples above the threshold at the highest dilution tested (1:13107200), the value 13107200 was used for the titer. Samples where a well value was the same as 50000 RLU were given the interpolated titer value at that exact dilution. If a sample crossed the 50000 RLU threshold between two dilutions, the titer at the crossing point was calculated by connecting the nearest points above and below the threshold, and solving for the dilution at which this line crossed the threshold.Serum IgG and Human GII.3 Monoclonal Antibody ELISA Assays

[0243] Maxisorp 384-well plates (Thermo Fisher, Waltham, MA) were coated with 50 ng / well of VLP diluted in Dulbecco’s phosphate-buffered saline (DPBS). Plates were sealed, centrifuged at 1500 xg for 1 minute and incubated overnight at 4 °C in a humidity chamber (21 °C, 80% RH). Human IgGl monoclonal antibodies w ere diluted in DPBS to a starting concentration of 200 pg / mL (antibody) and subsequently diluted in a four-fold, ten-point series in 3% nonfat dry’ milk powder (NFDMP) in PBS containing 0.1% tween-20 (PBS-T). Human monoclonal antibodies w ere discovered by panning against a human Fab phase display’ library with Norovirus VLPs as antigens. The dilution masterblock was sealed, centrifuged at 1500 x g for 1 minute and stored overnight at 4°C in a humidity chamber (21°C, 80% RH). The assay plates were washed 6x with IX PBS-T (100 pL / well) and blocked with 3% NFDMP in PBS-T (80 pL) for 30 min in a humidified incubator (21 °C, 80% RH). Monoclonal antibodies were added to 384 well assayplates and incubated for 2 hours in a humidified incubator to allow for VLP binding (21°C, 80% RH). Following incubation, the assay plates were washed 6x with PBS-T (100 pL / well).Antibody-bound VLP was detected with goat anti -human Ig-Fc fragment conjugated to HRP (Jackson ImmunoResearch, West Grove, PA) in 3% NFDMP in PBS-T (1:10,000) for 1 hour in a humidified incubator (21°C, 80% RH). Assay plates were washed 6x with PBS-T (100 pL / well) and developed with luminescent substrate (Pierce West Pico PLUS, ThermoFisher, Waltham, MA) for 15 min at RT. Ultrasensitive luminescence was read on an Envision plate reader (PerkinElmer, Waltham, MA) at 0.1 seconds per well. Interpolated serum antibody endpoint titers were calculated as the highest dilution where the relative light unit (RLU) signal is above a 50000 RLU cutoff. Samples where no dilution crossed the threshold were given a placeholder titer of “25”. For samples above the threshold at the highest dilution tested (1:13107200), the value 13107200 was used for the titer. Samples where a well value was the same as 50000 RLU were given the interpolated titer value at that exact dilution. If a sample crossed the 50000 RLU threshold between two dilutions, the titer at the crossing point was calculated by connecting the nearest points above and below the threshold, and solving for the dilution at which this line crossed the threshold.Serum IgG Antibody ELISA Assay (FIG.9A)

[0244] Maxisorp 384-well plates (Thermo Fisher, Waltham, MA) were coated with 50 ng / well of VLP diluted in Dulbecco’s phosphate-buffered saline (DPBS). Plates were sealed, centrifuged at 1500 xg for 1 minute and incubated overnight at 4°C in a humidity chamber (21 °C, 80% RH). Serum samples were diluted in DPBS to a starting concentration of 200 pg / mL (antibody) or 1 / 50 (serum) and subsequently diluted in a four-fold, ten-point series in 3% nonfat dry milk powder (NFDMP) in PBS containing 0.1% tween-20 (PBS-T). The dilution masterblock was sealed, centrifuged at 1500 x g for 1 minute and stored overnight at 4°C in a humidity7chamber (21°C, 80% RH). The assay plates were washed 6x with IX PBS-T (100 pL / well) and blocked with 3% NFDMP in PBS-T (80 pL) for 30 min in a humidified incubator (21°C, 80% RH). Serum was added to 384 well assay plates and incubated for 2 hours in a humidified incubator to allow for VLP binding (21°C, 80% RH). Following incubation, the assay plates were washed 6x with PBS-T (100 pL / well). Antibody-bound VLP was detected with goat anti-human Ig-Fc fragment conjugated to HRP (Jackson ImmunoResearch, West Grove, PA) in 3% NFDMP in PBS-T (1:10,000) for 1 hour in a humidified incubator (21°C, 80% RH). Assay plates were washed 6x with PBS-T (100 pL / well) and developed with luminescent substrate (Pierce West Pico PLUS, ThermoFisher, Waltham, MA) for 15 min at RT. Ultrasensitive luminescence was read on anEnvision plate reader (PerkinElmer, Waltham, MA) at 0.1 seconds per well. Interpolated serum antibody endpoint titers were calculated as the highest dilution where the relative light unit (RLU) signal is above a 50000 RLU cutoff. Samples where no dilution crossed the threshold were given a placeholder titer of “25”. For samples above the threshold at the highest dilution tested (1: 13107200), the value 13107200 was used for the titer. Samples where a well value was the same as 50000 RLU were given the interpolated titer value at that exact dilution. If a sample crossed the 50000 RLU threshold between two dilutions, the titer at the crossing point was calculated by connecting the nearest points above and below the threshold, and solving for the dilution at which this line crossed the threshold.HGBA Blocking Assay (FIG. 9B)

[0245] Maxisorp 384-well assay plates (Thermo Fisher, Scientific) were coated with human saliva (Precision for Medicine) diluted 1:500 in DPBS. Assay plates were centrifuged @ 1500 xg for 1 minute and incubated overnight at 4°C in a humidity chamber (21°C 80% RH). Ten, twofold serial dilutions of human sera (iProcess Global Research) were prepared in 5% Nonfat Powdered Dry Milk in Phosphate Buffered Saline with Tween 20 (NFPDM-PBSt) in a 384-well masterblock (Greiner) with a starting dilution of 1:10. The masterblock was sealed with aluminum foil and centrifuged @ 1500 x g for 1 minute. The plates were stored overnight at 4C in a humidity chamber. GII.3 wild type (WT) and GII.3 disulfide stabilized 1 (DS1) VLPs were prepared at 0.5pg / ml in a 1 : 1 dilution with human sera serial dilutions in 5% NFPDM PBS-T and pre-incubated for Ih in a humidified incubator (21°C, 80% RH) prior to addition to assay plates. The assay plates were rinsed 6X with PBS-T and blocked with 5% NFPDM PBS-T for 30 min in ahumidified incubator (21°C, 80% RH). VLP human sera premix were transferred to assay plates and incubated for 2h in a humidified incubator (21 °C, 80% RH). Assay plates were washed 6X with PBS-T. Binding of VLP to human saliva was detected with GII.3 specific polyclonal rabbit serum in 5% NFPDM PBS-T (1 :3000) for Ih in a humidified incubator (21°C, 80% RH). Assay plates were rinsed 6X with PBS-T. Goat anti rabbit-HRP(Fc)-IgG (1:5000 in 5% NFPDM) (Jackson ImmunoResearch) was added to assay plates and incubated for Ih in a humidified incubator (21 °C, 80% RH). Assay plates were washed 6X with PBS-T. Luminescent signal was developed for 15 min at RT with luminescent substrate (West Pico PLUS, Thermo Scientific, Pierce). BT50 values were defined as the titer at which luminescence readings were 50% of the positive control. A value of 10 was assigned to samples with a BT50 less than the starting dilution of 20. A blocking control serum sample was used as another control, with plates rejected26069if the BT50 for the blocking control was greater than one dilution above or below the known BT50.mRNA / LNP Generation

[0246] VP1 expressing mRNAs were generated by Trilink (San Diego, CA) with Nl-methyl-pseudouridine triphosphate modification and clean-cap. LNP encapsulating mRNA were prepared by rapid precipitation process as previously described (Gindy ME, Feuston B, Glass A, Arrington L, Haas RM, Schariter J, Stirdivant SM. 2014. Stabilization of Ostwald ripening in low molecular weight amino lipid nanoparticles for systemic delivery of siRNA therapeutics. Mol Pharm 11:4143-53). The lipid components of the LNP comprised an asymmetric ionizable amino lipid, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and poly (ethylene glycol)2000-dimyristoylglycerol (PEG2000-DMG) in a molar ratio of 58:30:10:2, respectively.Rabbit Polyclonal Sera Generation

[0247] Six female New Zealand white rabbits (approx, four months old at receipt) from Jackson Labs were housed singly and randomized into two groups. Animals were inoculated three times (two weeks between doses) intramuscularly in the quadriceps bilaterally. Inoculations consisted of 50 pg of appropriate virus-like particle (VLP) and 225 pg of amorphous aluminum hydroxyphosphate sulfate in 250 pl total (remaining volume was phosphate-buffered saline), administered once in each quadriceps at each time point. Blood draws were performed at weeks 1, 3, 5, and 6 after the first inoculation. All were from the ear artery, and the final was a terminal bleed. For all survival blood draws, animals were dosed with acepromazine (1-3 mg / kg subcutaneously) and approximately 2 ml of blood was drawn from the ear artery. For terminal bleeds, animals were administered ketamine (100 mg) and xylazine (20 mg) intramuscularly, then were bleed from the ear artery until no more blood could be acquired. A cardiac puncture was performed for remaining blood, and then euthanasia was performed by administration of Euthasol intravenously. Euthanasia was confirmed by cutting the diaphragm to cause pneumothorax. All blood was spun at 4000 rpm for 5 minutes to separate serum, which was transferred to new tubes.Mouse Immunizations

[0248] Mouse studies were approved by the Institutional Animal Care and Use Committee at Merck & Co., Inc. (Rahway, NJ, USA). BALB / c mice (CRL) aged 5-7 weeks were immunized26069with mRNAs in a lipid nanoparticle formulation. Two weeks following each immunization, blood was drawn for serological assays.Table of SequencesTable 4. Sequences260692606926069260692606926069

Claims

WHAT IS CLAIMED IS:

1. A synthetic polypeptide, comprising a VP1 polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 7, and 11, wherein at least one of the amino acid residues at positions 112 and 189 of SEQ ID NO: 1 is a cysteine.

2. The synthetic polypeptide of claim 1, wherein the amino acid residues at both positions 112 and 189 of SEQ ID NO: 1 are cysteines.

3. The synthetic polypeptide of claim 1 or claim 2, wherein the VP1 polypeptide sequence is at least 98% identical to any of SEQ ID NOs: 1, 7, and 11.

4. The synthetic polypeptide of claim 1, wherein the VP1 polypeptide sequence comprises any of SEQ ID NOs: 1, 7, and 11.

5. The synthetic polypeptide of any one of claims 1-3, wherein the VP1 polypeptide sequence is capable of self-assembly into non-infectious virus-like particles (VLPs).

6. A synthetic polypeptide comprising mutated VP 1 polypeptide comprising a sequence of amino acids as set forth in any of SEQ ID NOs: 2, 8, and 12, with the exception of a single amino acid substitution relative to the amino acid sequence in any of the foregoing sequences, wherein one of (i) amino acid position 112 and (ii) amino acid position 189. in any of SEQ ID NOs: 2, 8. and 12, comprises a cysteine residue.

7. The synthetic polypeptide of claim 6, wherein the mutated VP1 polypeptide is capable of self-assembly into non-infectious VLPs.

8. A composition, comprising the synthetic polypeptide of any one of claims 1-7 and a pharmaceutically acceptable carrier.

9. A synthetic polynucleotide, comprising a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 3, 9, and 13, wherein the nucleic acid sequence encodes the VP1 polypeptide of any one of claims 1-7.

10. The synthetic polynucleotide of claim 9, wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 3, 9, and 13.

11. The synthetic polynucleotide of claim 9 or claim 10, wherein the synthetic VP1 polypeptide is capable of self-assembly into non-infectious virus-like particles.

12. A synthetic polynucleotide, comprising a messenger ribonucleic acid (mRNA) sequence that is at least 95% identical to SEQ ID NO: 5, wherein the mRNA encodes the VP1 polypeptide of any one of claims 1-7.

13. The synthetic polynucleotide of claim 12, wherein the mRNA comprises SEQ ID NO: 5.

14. The synthetic polynucleotide of claim 12 or claim 13, wherein the VP1 polypeptide comprises SEQ ID NO: 1.

15. The synthetic polynucleotide of any one of claims 12-14, wherein at least one uridine in the mRNA sequence is replaced with an N1 -methylpseudouridine.

16. The synthetic polynucleotide of claim 15, wherein each of the uridines in the mRNA sequence is replaced with an N1 -methylpseudouridine.

17. The synthetic polynucleotide of any one of claims 12-16, wherein the synthetic VP1 polypeptide is capable of self-assembly into non-infectious virus-like particles (VLPs).

18. A composition, comprising the synthetic polynucleotide of any one of claims 9-17 and a pharmaceutically acceptable carrier.

19. A composition, comprising any one of the synthetic polynucleotides of claims 12-17, wherein the synthetic polynucleotide is encapsulated in a lipid nanoparticle (LNP).

20. The composition of claim 19, wherein the LNP comprises a cationic lipid, a sterol, a phospholipid, and a polyethyleneglycol-lipid.

21. The composition of claim 20, wherein the cationic lipid is selected from the group consisting of:

22. A vector comprising the synthetic polynucleotide of any one of claims 9-11.

23. A non-infectious virus-like particle (VLP), comprising a synthetic polypeptide of any one of claims 1-7.

24. A host cell comprising any of (i) the synthetic polypeptides of any one of claims 1-7, (ii) the synthetic polynucleotides of any one of claims 9-17, (iii) the compositions of any one of claims 8 and 18-21, (iv) the vector of claim 22, or (v) the non-infectious VLP of claim 23.2606925. A norovirus vaccine, comprising the polynucleotide of any one of claims 12-17 and a pharmaceutically acceptable carrier.

26. The norovirus vaccine of claim 25, further comprising an adjuvant.

27. A method of treating or preventing a disorder related to a norovirus infection in a subject in need thereof, comprising: administering to the subject an effective amount of any of (i) the synthetic polypeptides of any one of claims 1-7, (ii) the synthetic polynucleotides of any one of claims 9-17, (iii) the compositions of any one of claims 8 and 18-21, (iv) the vector of claim 22, (v) the non-infectious VLP of claim 23, or (vi) the norovirus vaccine of claim 25 or claim 26.

28. A method of treating or preventing a disorder related to a norovirus infection in a subject in need thereof, comprising: administering to the subject an effective amount of (i) the synthetic polynucleotides of any one of claims 9-17, (ii) the compositions of any one of claims 18-21, (iii) the vector of claim 22, or (iv) the norovirus vaccine of claim 25 or claim 26.

29. A method of reducing the likelihood of a norovirus infection or a disorder associated with said infection in a subject in need thereof, comprising: administering to the subject an effective amount of (i) the synthetic polypeptides of any one of claims 1-7, (ii) the synthetic polynucleotides of any one of claims 9-17, (iii) the compositions of any one of claims 8 and 18-21, (iv) the vector of claim 22, (v) the non-infectious VLP of claim 23, or (vi) the norovirus vaccine of claim 25 or claim 26.

30. A method of inducing an immune response against norovirus GII.3 in a subject in need thereof, comprising: administering to the subject an effective amount of (i) the synthetic polynucleotides of any one of claims 9-17, (ii) the compositions of any one of claims 18-21, (iii) the vector of claim 22, or (iv) the norovirus vaccine of claim 25 or claim 26.

31. A method of making norovirus GII.3 virus-like particles (VLPs), comprising: (i) expressing a synthetic polynucleotide of any one of claims 9-17 in an expression system under conditions suitable for expression of the synthetic polynucleotide to produce a VP1 polypeptide, (ii) allowing the VP1 polypeptide to self-assemble into norovirus GII.3 VLPs and (iii) optionally isolating the VLPs.2606932. Use of (i) the synthetic polypeptides of any one of claims 1-7, (ii) the synthetic polynucleotides of any one of claims 9-17, (iii) the compositions of any one of claims 8 and 18-21, (iv) the vector of claim 22, (v) the non-infectious VLP of claim 23, or (vi) the norovirus vaccine of claim 25 or claim 26, to induce an immune response against norovirus GII.3 in a subject in need thereof.

33. Use of (i) the synthetic polypeptides of any one of claims 1-7, (ii) the synthetic polynucleotides of any one of claims 9-17, (iii) the compositions of any one of claims 8 and 18-21. (iv) the vector of claim 22. (v) the non-infectious VLP of claim 23, or (vi) the norovirus vaccine of claim 25 or claim 26, to reduce the likelihood of infection with norovirus GII.3 in a subject in need thereof, or a disorder associated with said infection.

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