WO2024254552A1 - Stabilized flavivirus vaccines - Google Patents

Stabilized flavivirus vaccines Download PDF

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WO2024254552A1
WO2024254552A1 PCT/US2024/033144 US2024033144W WO2024254552A1 WO 2024254552 A1 WO2024254552 A1 WO 2024254552A1 US 2024033144 W US2024033144 W US 2024033144W WO 2024254552 A1 WO2024254552 A1 WO 2024254552A1
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protein
seq
amino acid
positions
protein comprises
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PCT/US2024/033144
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Arvind Sharma
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Modernatx, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Flavivirus is a genus of positive-strand RNA viruses in the family Flaviviridae.
  • the genus includes the West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus, Japanese encephalitis virus, Powassan virus, St. Louis Encephalitis virus, Usutu virus, and Spondweni virus which may cause disease (e.g., encephalitis) in humans.
  • Dengue virus is a mosquito-borne flavivirus that causes dengue fever, a severe flu-like illness. There are four distinct, but closely related, serotypes of the virus that cause dengue (DENV-1, DENV-2, DENV-3, and DENV-4).
  • Dengue virus is transmitted to humans through the bites of infected female mosquitoes, primarily the Aedes aegypti mosquito. This species prefers to bite humans and is most active during daylight hours. After virus incubation for about a week, an infected human may develop dengue fever. Dengue fever is characterized by high fever, severe headache, pain behind the eyes, joint and muscle pain, rash, and mild bleeding (such as nose or gum bleed, or easy bruising). In severe cases, it can evolve into severe dengue or dengue hemorrhagic fever, which can cause bleeding, blood plasma leakage, and low platelet count.
  • the disease is endemic in more than 100 countries in the World Health Organization's (WHO) regions of Africa, the Americas, the Eastern Mediterranean, Southeast Asia, and the Western Pacific.
  • WHO World Health Organization's
  • DENGVAXIA ® which is a live, tetravalent vaccine; however, this vaccine is approved by the FDA only for use in individuals 9 through 16 years of age with laboratory-confirmed previous dengue infection and living in endemic areas.
  • SUMMARY Flaviviruses are pathogens that cause severe morbidity and mortality in humans. These viruses cycle between the arthropod (mosquito or tick) and vertebrate hosts.
  • Flaviviruses are structurally similar to one another (see, e.g., FIG.1A, which illustrates structural similarities between DENV1, DENV2, DENV3, DENV4, and ZIKV).
  • DENVs dengue virus serotypes
  • ZIKV dengue virus serotypes
  • the envelope (E) protein dimer in its prefusion conformation is a metastable protein that transforms into the stable post-fusion trimeric form upon exposure to an acidic pH.
  • DENVs have evolved to produce immature particles as decoy antigens that have the outer shell made of (M/E) heterodimers arranged as trimeric spikes with a viral chaperone protein, pr, on the tip of E protein and covalently linked to M protein thus forming 60 (prM/E) 3 spikes on the surface.
  • VLPs non-infectious Virus-Like Particles
  • the engineered heterotetramers are stabilized in a native conformation in order to elicit an efficacious protective immune response.
  • the use of such heterotetramers is unique.
  • Previous vaccine designs have relied on structures that do not closely approximate the three-dimensional structure of native flaviviruses and, therefore, are not optimal immunogens.
  • the disclosure in some aspects, provides a stabilized flavivirus heterotetramer, the heterotetramer comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein the E protein comprises an amino acid substitution at each of two positions, wherein the two positions correspond with position 76 and position 107 of SEQ ID NO: 276, and the M protein comprises a N-terminus and a C-terminus; wherein the N-terminus comprises an N-terminal region comprising at least one amino acid substitution relative to a corresponding wild-type flavivirus M protein.
  • E flavivirus envelope
  • M flavivirus membrane
  • the shared confirmational elements of the flaviviruses allow one of skill in the art to determine those amino acid positions from the related viruses, which correspond to the reference amino acid positions in the Dengue 2 amino acid sequence (see FIG. 1A). Those amino acid positions are amenable to mutation in order to achieve the same stabilizing effect and still allow for the generation of antibodies that will recognize the native flavivirus and, therefore, be protective. It will be understood that in the subsequent paragraphs, the recitation of amino acid positions (e.g., 107 and 76) embraces the corresponding amino acid positions in related flaviviruses which correspond to those positions.
  • the amino acid substitution at position 107 comprises a substitution with a charged bulky residue.
  • the amino acid substitution at position 76 comprises a substitution with a charged bulky residue.
  • the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R).
  • the at least one substitution within the N-terminal region is selected from the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105; wherein the positions correspond with positions 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105 of SEQ ID NO: 275.
  • the at least one substitution within the N- terminal region is selected from the following substitutions: (a) the amino acid substitution at position 90 is valine (V) or isoleucine (I); (b) the amino acid substitution at position 91 is isoleucine (I) or cysteine (C); (c) the amino acid substitution at position 93 is isoleucine (I), phenylalanine (F), tyrosine (Y), or tryptophan (W); (d) the amino acid substitution at position 94 is isoleucine (I), leucine (L), asparagine (N), or lysine (K); (e) the amino acid substitution at position 96 is cysteine (C); (f) the amino acid substitution at position 97 is isoleucine (I) or cysteine (C); (g) the amino acid substitution at position 99 is isoleucine (I) or leucine (L); (h) the amino acid substitution at position 101 is methionine (M), isoleu
  • the E protein comprises an amino acid substitution at each of two positions, wherein the two positions correspond with position 76 and position 107 of SEQ ID NO: 276, and the E protein comprises the wild-type amino acid at position 106, wherein the position corresponds with position 106 of SEQ ID NO: 276.
  • the E protein further comprises at least one additional substitution selected from the group consisting of: an amino acid substitution at position 131; an amino acid substitution at position 194; and an amino acid substitution at position 134; wherein the positions correspond with positions 131, 194, and 134 of SEQ ID NO: 276.
  • the at least one additional substitution is selected from the following amino acid substitutions: (a) the amino acid substitution at position 131 is alanine (A), asparagine (N), or leucine (L); (b) the amino acid substitution at position 194 is aspartic acid (D); and (c) the amino acid substitution at position 134 was asparagine (N) or aspartic acid (D); wherein the positions correspond with positions 131, 194, and 134 of SEQ ID NO: 276.
  • the E protein and the M protein comprise a fusion protein, the fusion protein comprising, from N-terminus to C-terminus, the M protein and the E protein.
  • the E protein and the M protein comprise a fusion protein, the fusion protein comprising, from N-terminus to C-terminus, the E protein and the M protein and optionally wherein a linker is positioned between the E protein and the M protein, further optionally wherein the linker is a flexible linker.
  • the linker comprises GGGG (SEQ ID NO: 364) or GGPG (SEQ ID NO: 365).
  • the E protein further comprises an amino acid substitution at position 495, wherein the position corresponds with position 495 of SEQ ID NO: 276; and/or the M protein further comprises an amino acid substitution at position 90, wherein the position corresponds with position 90 of SEQ ID NO: 275.
  • the amino acid substitution of the E protein is a glycine (G) at position 495, wherein the position corresponds with position 495 of SEQ ID NO: 276; and/or wherein the amino acid substitution of the M protein is a glycine (G) at position 90, wherein the position corresponds with position 90 of SEQ ID NO: 275.
  • the heterotetramer further comprises a cavity created by: amino acid substitutions at positions 262 and 265 of the E protein, wherein the positions correspond with positions 262 and 265 of SEQ ID NO: 276; and amino acid substitutions at position 97 of the M protein, wherein the position corresponds with position 97 of SEQ ID NO: 275.
  • the amino acid substitutions of positions 262 and 265 of the E protein are each an alanine (A) or serine (S), wherein the positions correspond to positions262 and 265 if SEQ ID NO: 276; and the amino acid substitution of position 97 of the M protein is alanine (A), wherein the position corresponds to position 97 of SEQ ID NO: 275.
  • the E protein further comprises the following amino acid substitutions: a tyrosine (Y), glutamine (Q), or asparagine (N) at position 267; and a lysine (K) or arginine (R) at position 445; wherein the positions correspond to position 267 and 445 of SEQ ID NO: 276.
  • the N-terminal region of the M protein comprises at least one amino acid substitution of a non-cysteine amino acid with a cysteine amino acid, relative to the corresponding wild-type flavivirus M protein.
  • the heterotetramer further comprises a signal peptide.
  • the signal peptide comprises an amino acid sequence selected from SEQ ID NOs: 271 or 272.
  • the flavivirus is selected from the group consisting of: Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus and Omsk virus.
  • the flavivirus is Dengue virus. In some embodiments, the flavivirus is Zika virus. In some embodiments, the Zika virus E protein further comprises an introduced glycosylation site. In some embodiments, the introduced glycosylation site comprises an N-X-S motif in positions 67-69, wherein the positions correspond to positions 67-69 of SEQ ID NO: 282. In one embodiment, the disclosure pertains to a nucleic acid molecule encoding a stabilized heterotetramer comprising an E protein and M protein of a flavivirus as described herein.
  • the disclosure in some aspects and embodiments, provides a DNA plasmid comprising a nucleic acid encoding the E protein and M protein capable of forming any one of the stabilized flavivirus heterotetramers described herein.
  • the disclosure in some aspects and embodiments, provides a messenger ribonucleic acid (mRNA) vaccine comprising an mRNA polynucleotide having an open reading frame (ORF) encoding a single chain protein comprising the E protein and M protein capable of forming any one of the stabilized flavivirus heterotetramers described herein.
  • mRNA messenger ribonucleic acid
  • ORF open reading frame
  • the mRNA vaccine comprises: (A) a first mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NOs: 112, 217, 335-340, and 353-354; (B) a second mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 27, 195, 302-319, 328-334, and 358-360; (C) a third mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 117, 175, 320-327, 341-343, and 361-363; and (D) a fourth mRNA polynucleotide comprising an ORF encoding a
  • the mRNA further comprises: (E) a fifth mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 108, 140-142, 177, 285-301, and 347-352.
  • the mRNA is formulated in a lipid nanoparticle.
  • the disclosure provides a composition comprising at least one of the mRNA described herein and a lipid nanoparticle.
  • the lipid nanoparticle comprises 40-55 mol% of a lipid of Compound 1 or A7, 30-45 mol% cholesterol, 5-15 mol% 1,2 distearoyl-sn-glycero-3- phosphocholine (DSPC), and 1-5 mol% PEG-modified lipid.
  • the lipid nanoparticle comprises Compound 1:
  • the lipid nanoparticle comprises A7:
  • the PEG-modified lipid is PEG2000-DMG.
  • the mRNA comprises a chemical modification, optionally selected from the group consisting of: pseudouridine ( ⁇ ), 1-methyl-pseudouridine (m1 ⁇ ), 1-ethylpseudouridine (e1 ⁇ ), 5- methoxyuridine (mo5U), 5-methylcytidine (m5C), 5-methoxymethyluridine, 5- methylthiouridine, 1-methoxymethylpseudouridine, 5-methoxycytidine, and any combination thereof.
  • the mRNA comprises 1-methyl-pseudouridine.
  • the ORF comprises 1-methyl-pseudouridine, adenosine, guanosine, and cytosine.
  • the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject one or more doses of the any one of the stabilized flavivirus heterotetramers described herein in protein or nucleic acid form, e.g., any one of the DNA, e.g., plasmid DNA described herein, any one of the mRNA described herein, or any one of the compositions described herein in an effective amount to produce an immune response.
  • the any one of the stabilized flavivirus heterotetramers described herein in protein or nucleic acid form e.g., any one of the DNA, e.g., plasmid DNA described herein, any one of the mRNA described herein, or any one of the compositions described herein in an effective amount to produce an immune response.
  • the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject one or more doses of the any one of the stabilized flavivirus heterotetramers described herein, any one of the DNA, e.g., plasmid DNA described herein, any one of the mRNA described herein, or any one of the compositions described herein in an amount effective at inducing in the subject a population of neutralizing antibodies that cross reacts with a naturally occurring flavivirus.
  • the mRNA vaccine is administered intramuscularly.
  • the flavivirus is Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus, Omsk virus, or any combination thereof.
  • the flavivirus is Dengue virus.
  • the flavivirus is Zika virus.
  • the disclosure in some aspects, provides a method of inducing neutralizing antibodies in a subject by administering a stabilized flavivirus heterotetramer comprising: means for inducing an immune response to the flavivirus in the subject; and a flavivirus envelope (E) protein, wherein the E protein comprises an amino acid substitution at each of position 76 and at position 107, wherein the positions correspond to SEQ ID NO: 276; and a flavivirus membrane (M) protein, wherein administration of the flavivirus heterotetramer has increased neutralization antibody titers compared to wild-type flavivirus prME protein.
  • E flavivirus envelope
  • M flavivirus membrane
  • FIG.1A is a sequence alignment between four dengue virus serotypes and Zika virus.
  • the top panel shows the sequence alignment between prM proteins (rows 1-2; DENV1, SEQ ID NO: 273 A; DENV2, SEQ ID NO: 275; DENV3, SEQ ID NO: 277; DENV4, SEQ ID NO: 279; ZIKV, SEQ ID NO: 281).
  • the gray arrow indicates the furin cleavage site between pr and M proteins.
  • the lower panel shows the sequence alignment between E proteins from five viruses (rows 3-8; DENV1, SEQ ID NO: 274; DENV2, SEQ ID NO: 276; DENV3, SEQ ID NO: 278; DENV4, SEQ ID NO: 280; ZIKV, SEQ ID NO: 282).
  • FIGs.1B and 1C illustrate the structure of a flavivirus (M/E) 2 heterotetramer from a virion.
  • the flavivirus E protein ectodomain consists of three domains (EDI, EDII, and EDIII) followed by a transmembrane region labelled as “stem” in the figure.
  • the E protein interacts with the transmembrane protein (M) that provides a scaffold and curvature to the E protein dimer.
  • FIG.1B shows the top view of (M/E) 2 heterotetramer domain (top panel).
  • FIG.1C provides an illustration of the stem and M protein scaffold and E dimer (top panel) and an open book view of the same (bottom panel).
  • the antigens described herein are unique because they comprise heterotetramer structures, which stabilize the antigen in a prefusion conformation and lead to a stronger immune response than E-dimers or wild-type proteins.
  • FIG.2 shows electron microscopy analysis of virus-like particles (VLPs) formed using engineered proteins described herein. Negative stain electron microscopy and cryogenic (cryo- EM) imaging were used.
  • DENV2 102 + Fab C8 indicates DENV2-M/E_102 VLPs in complex with Fab fragments of bnAb C8.
  • ZIKV 50 + Fab C8 indicates ZIKV_50_E/M VLPs in complex with Fab fragments of bnAb C8.
  • the box in each panel highlights the VLPs. VLPs of sizes 40 to 60 nm were observed for each construct.
  • FIG.3 shows an SDS-PAGE gel of ZIKV_HA_50_E/M_VLPs following purification, demonstrating the presence of covalent (EM)2 heterotetramer VLPs.
  • Expi293 cells were transfected using plasmid containing a ZIKV_HA_50_E/M gene. The supernatant was harvested after 72 hours and the VLPs were purified using sucrose gradient method as described previously (Shen et al., eLife 7:e38970). These partially purified VLPs were further purified by affinity chromatography using Fab C8 as bait. Purified samples were run on SDS-PAGE in reducing (+ ⁇ ME) and non-reducing conditions (- ⁇ ME). M; molecular weight marker.
  • E and M proteins are a single chain and the engineered (EM) 2 heterotetramer is covalently stabilized and thus in non-reducing conditions this protein runs at a molecular weight of ⁇ 130 kDa.
  • the Fab C8 band can be seen at approximately 50 kDa, which also contains the covalently linked heavy and light chains.
  • the EM single chain protein was observed at a molecular weight of approximately 65 kDa (M protein ⁇ 8.5 kDa + E protein ⁇ 55 kDa).
  • Fab C8 was also reduced into heavy chain (HC) and light chain (LC) bands. The first two lanes show Fab C8 in non-reducing and reducing conditions, respectively.
  • FIGs.4A-4B show the relative abundance of intracellular and surface proteins (FIG.4A) and of soluble fractions (VLPs) (FIG.4B) following monovalent transfection (left graphs) and following tetravalent transfection (right graphs).
  • AS indicates exemplary designed proteins (V1 candidates) and WT indicates wild type proteins of 4 exemplary Dengue viruses. Briefly, 72 hours post-transfection in 24 well plate, 100 ul of cell suspension was taken out and pelleted using by centrifugation at 500g for 5 min.
  • FIG.4A pelleted cells were washed with PBS and subjected to mass-spectrometry evaluation for quantifying the relative expression of designs vs WT. High relative abundance for designs was observed compared to the WT.
  • FIG.4B shows the supernatant tested for secreted VLP/ protein presence using mass-spectrometry after centrifugation. The relative peptide count between the design and WT protein was determined, for e.g. in monovalent transfection, there was approximately a 40000 higher peptide count for AS D1 compared to the WT D1.
  • FIG.5 shows electron microscopy analysis of (ME)2 stabilized VLPs engineered VLPs using negative stain electron microscopy and cryogenic (cryo-EM) imaging.
  • FIG.6 shows an SDS-PAGE gel of DENV VLPs (V2 designs) following purification as described in FIG.3, demonstrating the presence of covalent (EM)2 heterotetramers in Expi203 cells.
  • FIGs.7A-7D show flow cytometry results demonstrating selected DENV antigen (V1 designs) binding to specific antibodies. Each antigen, designed (denoted as “Stab”, right bar) or wild-type (WT, left bar), was tested individually (DENV1, DENV2, DENV3, DENV4) and together (“All 4”).
  • the percent of positive cells (top row) and the mean fluorescence intensity (bottom row) are shown.
  • Cell samples were prepared as described in FIG.4A.
  • A11; mAb EDE2 A11, G-A11; a germline version of the EDE2 mAb A11, B7; mAb EDE2 B7, C8, mAb EDE1 C8, C10; mAb EDE1 C10, G-C10; a germline version of the EDE1 mAb C10, and mAb SlgN3C are the bnAbs that cross-react with all four DENVs to varying extent.
  • FIG.8 is four graphs showing monovalent homologous antibody responses (neutralization titers) in vivo.
  • mice were immunized with the LNPs containing mRNA that encoded for wild type (WT) DENVs1-4 VLPs or the (ME)2 stabilized VLPs (V1).
  • PBS no LNPs were injected - sterile PBS used instead, WT 1; 1 ug of LNPs encoding WT DENV VLP were injected.
  • V14; 4 ug of the V1 encoding LNPs were injected.
  • a prime (day 1) boost (day 22) regime was followed and blood samples were collected from mice on day 57.
  • FIGs.9A-9D show cross-reactive antibody responses (neutralization titers) following DENV1 vaccination (FIG.9A), DENV2 vaccination (FIG.9B), DENV3 vaccination (FIG.9C), and DENV4 vaccination (FIG.9D). Control is WT samples.
  • FIG.10 is four graphs showing serotype-specific responses following administration of the tetravalent composition (measured by neutralization titer).
  • FIG.11 shows the immunological balance across the four DENV serotypes at the 1 ⁇ g (left graph) and 4 ⁇ g (right graph) doses.
  • DETAILED DESCRIPTION Flaviviruses are icosahedral structures having a lipid envelope covered densely with surface projections composed of multiple copies of M (membrane) and multiple copies of E (envelope) glycoproteins. The E glycoproteins are organized as dimers, paired horizontally head to tail, on the virion surface. Flaviviruses are approximately 50 nm spherical particles that, in their mature, infectious form have an outer shell made of 90 membrane (M) protein and envelope (E) protein heterotetramers (M/E)2 arranged in a herringbone pattern.
  • M membrane
  • E envelope
  • M/E protein heterotetramers
  • This (M/E)2 outer shell is the main target of the neutralizing antibody response and cross-reactivity among flaviviruses has been reported as the M and E proteins are not only structurally conserved among these viruses but can also have a high sequence identity of > 50% (Rey et al., Cell, 172(6), 1319- 1334, 2018; Duquerroy et al., Encyclopedia of Virol.1, 290-302, 2021) (FIG.1A). While the immune response against the prefusion (M/E) 2 heteroterameric form is protective in nature, the response against immature form is often non-neutralizing and can lead to antibody-dependent enhancement (ADE) of the disease.
  • ADE antibody-dependent enhancement
  • bnAbs broadly neutralizing antibodies
  • These protective antibodies target the mature shell of virus particles and lock the outer shell in a pre-fusion conformation.
  • the only known class of bnAbs that targets all four DENVs and ZIKV is called anti-E dimer epitope antibodies (Fey et al., EMBO Rep.2018 Feb; 19(2):206-224; Dejnirattisai et al., Nature Immunology, 16, 170-177; Sharma et al., Cell.2021 Dec 9;184(25):6052-6066).
  • VLPs engineered non-infectious Virus-Like Particles
  • FIG.1A shows a sequence alignment between the four DENV serotypes and ZIKV, where exemplary positions mutated for stabilization are boxed.
  • This sequence alignment is based on the conformational similarity among these viruses and the numbering of positions amenable to mutation in a reference sequence (e.g., Dengue 2) can be used to determine those positions in related sequences that are mutated to achieve the same result.
  • a reference sequence e.g., Dengue 2
  • mutations in the bc loop and fusion loop of the E protein, as well as the N-terminal of the M protein were found to stabilize the E protein in its prefusion conformation.
  • the structure-guided antigen designs were applied to multiple different flavivirus, demonstrating the applicability of the substitutions to flavivirus due to the high structural similarity between the flaviviruses.
  • Stabilized heterotetramers were designed for DENV (all four serotypes) and ZIKV and were then tested for expression and presence of the prefusion conformation heterotetramer. Additionally, engineered VLPs for yellow fever virus (YFV) designed using the structure-guided approach described herein showed improved expression and presence of the prefusion heterotetramer conformation.
  • Stabilized Flavivirus Heterotetramers Described herein are engineered flavivirus antigens comprising at least one amino acid substitution relative to their respective wild-type proteins and which form stabilized heterotetramers.
  • a stabilized flavivirus heterotetramer refers to a complex of four flavivirus proteins, including two E proteins and two M proteins having amino acid substitutions relative to wild-type E and M proteins.
  • the amino acid substitutions stabilize the complex of four proteins in a prefusion heterotetramer conformation.
  • stabilizing the heterotetramer in its prefusion conformation elicits an optimal immune response because the native curvature of the E protein is preserved.
  • each of the two E proteins includes at least a substitution at positions 76 and 107 (corresponding to a wild-type DENV2 E protein; SEQ ID NO: 276).
  • Position 76 of the DENV2 E protein is located in the bc loop region of the E protein.
  • the bc loop is a hydrophobic loop found in the second domain of the E protein and is linked to the Fu loop via a disulfide bond.
  • Position 107 of the DENV2 E protein is located in the fusion loop region of the E protein.
  • the fusion loop of the E protein forms the tip of the second domain of the E protein and is required for the low-pH- driven membrane fusion of the viral membrane with a host endosomal membrane.
  • the corresponding positions e.g., positions 76 and 107 of the wild-type DENV2 E protein
  • FIG.1A illustrates the analogous regions of DENV1-4 and ZIKV).
  • a substitution at position 76 of DENV2 corresponds to a substitution at position 76 of DENV1, DENV3, and DENV4, and position 77 of ZIKV, for example.
  • the numbered positions refer to DENV2 E and M proteins; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity.
  • the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid (e.g., SEQ ID NO: 276) with an amino acid comprising a charged bulky residue.
  • the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R).
  • the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with aspartic acid (D).
  • the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with glutamic acid (E).
  • the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with asparagine (N).
  • the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with glutamine (Q). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with lysine (K). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with tyrosine (Y). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with arginine (R).
  • the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid (e.g., SEQ ID NO: 276) with an amino acid comprising a charged bulky residue.
  • the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R).
  • the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with aspartic acid (D).
  • the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with glutamic acid (E). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with asparagine (N). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with glutamine (Q). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with lysine (K). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with tyrosine (Y).
  • the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with arginine (R).
  • the E protein comprises an amino acid substitution at each of positions 76 and 107 and does not comprise a mutation (e.g., amino acid substitution) at position 106 (for example, when the position corresponds to SEQ ID NO: 276).
  • the E protein comprises an amino acid substitution at each of positions 76 and 107 and a wild- type amino acid at position 106 (for example, when the position corresponds to SEQ ID NO: 276).
  • the E protein comprises a further amino acid substitution.
  • the further amino acid substitution in some embodiments, is located at the inter-dimer interface of the resulting VLP (e.g., corresponding to SEQ ID NO: 276).
  • the numbered positions refer to DENV2 E protein; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity.
  • the further amino acid substitution comprises an amino acid substitution at position 131.
  • the amino acid substitution at position 131 is selected from the group consisting of: alanine (A), asparagine (N), and leucine (L).
  • the amino acid substitution at position 131 is alanine (A).
  • the amino acid substitution at position 131 is asparagine (N). In some embodiments, the amino acid substitution at position 131 is leucine (L). In some embodiments, the amino acid substitution at position 194 is aspartic acid (D). In some embodiments, the amino acid substitution at position 134 is asparagine (N) or aspartic acid (D). In some embodiments, the amino acid substitution at position 134 is asparagine (N). In some embodiments, the amino acid substitution at position 134 is aspartic acid (D). In some embodiments the E protein comprises an amino acid substitution at position 131, at position 134, and at position 194.
  • the further E protein amino substitutions may comprise, in positions 131, 134, and 194 any of the following combinations: AND, ADD, NND, NDD, LND, or LDD.
  • the E protein comprises further amino acid substitution in order to stabilize its interactions with its stem region.
  • the numbered positions refer to DENV2 E protein; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity.
  • the E protein comprises an amino acid substitution at position 267 and/or 445 corresponding to SEQ ID NO: 276.
  • the E protein amino acid mutation is a tyrosine (Y), a glutamine (Q), or an asparagine (N) at position 267.
  • the E protein amino acid mutation is a lysine (K) or an arginine (R) at position 445.
  • the E protein comprises, in position 267 and 445, any of the following combinations: YK, YR, QK, QR, NK, or NR.
  • K lysine
  • R arginine
  • the E protein comprises, in position 267 and 445, any of the following combinations: YK, YR, QK, QR, NK, or NR.
  • the at least one amino acid substitution within the N-terminal region on the M protein is located within the N-terminal 38 amino acids (e.g., corresponding to SEQ ID NO: 275).
  • the numbered positions refer to DENV2 M protein; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity.
  • the at least one amino substitution within the N-terminal region of the M protein is selected from the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105.
  • the at least one amino acid substitution within the N-terminal region of the M protein comprises an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105.
  • the M protein comprises an amino acid substitution at position 90.
  • the M protein comprises an amino acid substitution at position 91.
  • the M protein comprises an amino acid substitution at position 93.
  • the M protein comprises an amino acid substitution at position 94.
  • the M protein comprises an amino acid substitution at position 96.
  • the M protein comprises an amino acid substitution at position 97.
  • the M protein comprises an amino acid substitution at position 99. In some embodiments, the M protein comprises an amino acid substitution at position 101. In some embodiments, the M protein comprises an amino acid substitution at position 103. In some embodiments, the M protein comprises an amino acid substitution at position 104. In some embodiments, the M protein comprises an amino acid substitution at position 105. In some embodiments, the amino acid substitution at position 90 of the M protein is valine (V). In some embodiments, the amino acid substitution at position 90 of the M protein isoleucine (I). In some embodiments, the amino acid substitution at position 91 of the M protein (e.g., corresponding to SEQ ID NO: 275) is cysteine (C).
  • the amino acid substitution at position 91 of the M protein isoleucine (I).
  • the amino acid substitution at position 91 of the M protein i.e., cysteine
  • C cysteine
  • the amino acid substitution at position 93 of the M protein is phenylalanine (F).
  • the amino acid substitution at position 93 of the M protein is tyrosine (Y).
  • the amino acid substitution at position 93 of the M protein is tryptophan (W).
  • the amino acid substitution at position 94 of the M protein (e.g., corresponding to SEQ ID NO: 275) is isoleucine (I). In some embodiments, the amino acid substitution at position 94 of the M protein is leucine (L). In some embodiments, the amino acid substitution at position 94 of the M protein is asparagine (N). In some embodiments, the amino acid substitution at position 94 of the M protein is lysine (K). In some embodiments, the amino acid substitution at position 96 of the M protein (e.g., corresponding to SEQ ID NO: 275) is cysteine (C).
  • the amino acid substitution at position 96 of the M protein is paired with an amino acid substitution of cysteine (C) at position 267 of the E protein, to form a disulfide bond.
  • the amino acid substitution at position 97 of the M protein e.g., corresponding to SEQ ID NO: 275
  • the amino acid substitution at position 97 of the M protein is cysteine (C).
  • the amino acid substitution at position 97 of the M protein i.e., cysteine
  • the amino acid substitution at position 99 of the M protein is isoleucine (I). In some embodiments, the amino acid substitution at position 99 of the M protein is leucine (L). In some embodiments, the amino acid substitution at position 101 of the M protein (e.g., corresponding to SEQ ID NO: 275) is methionine (M). In some embodiments, the amino acid substitution at position 101 of the M protein is isoleucine (I). In some embodiments, the amino acid substitution at position 101 of the M protein is phenylalanine (F).
  • the amino acid substitution at position 103 of the M protein (e.g., corresponding to SEQ ID NO: 275) is glycine (G). In some embodiments, the amino acid substitution at position 103 of the M protein is alanine (A). In some embodiments, the amino acid substitution at position 103 of the M protein is serine (S). In some embodiments, the amino acid substitution at position 103 of the M protein is cysteine (C). In some embodiments, the amino acid substitution at position 103 of the M protein (i.e., cysteine) is paired with an amino acid substitution of cysteine (C) at position 280 of the E protein, to form a disulfide bond.
  • G glycine
  • the amino acid substitution at position 103 of the M protein is alanine (A). In some embodiments, the amino acid substitution at position 103 of the M protein is serine (S). In some embodiments, the amino acid substitution at position 103 of the M protein is cysteine (C). In some embodiments, the amino acid
  • the amino acid substitution at position 104 of the M protein is serine (S).
  • the amino acid substitution at position 105 of the M protein is cysteine (C).
  • the amino acid substitution at position 105 of the M protein i.e., cysteine
  • the E protein and the M protein comprise a fusion protein.
  • the fusion protein comprises, from N-terminus to C-terminus, the M protein and the E protein.
  • the fusion protein comprises, from N-terminus to C-terminus, the E protein and the M protein.
  • a linker is positioned between the E protein and the M protein (i.e., from N-terminus to C-terminus, the fusion protein comprises the E protein, the linker, and the M protein).
  • the linker is a flexible linker. Without wishing to be bound by theory, it is thought that the inclusion of the flexible linker facilitates assembly of the heterotetramer and VLPs.
  • the linker comprises, or consists of, GGGG (SEQ ID NO: 364).
  • the linker comprises, or consists of, GGPG (SEQ ID NO: 365).
  • further amino acid substitutions are introduced to increase the flexibility of the linker region.
  • the further amino acid substitutions comprise an amino acid substitution at position 495 of the E protein and an amino acid substitution at position 90 of the M protein.
  • the further amino acid substitutions comprise a glycine (G) at position 495 of the E protein and a glycine (G) at position 90 of the M protein.
  • amino acid substitutions were used to generate a cavity between the E protein and the M protein to facilitate entry of the linker.
  • the cavity is introduced by amino acid substitutions at positions 262 and 265 of the E protein and position 97 of the M protein.
  • the amino acid substitutions comprise an alanine (A) or a serine (S) at each of position 262 and position 265 of the E protein; and an alanine (A) at position 97 of the M protein.
  • the tables below present a number of different engineered flavivirus fusion proteins.
  • the antigens for VLPs and the Pr protein sequence is followed by the M protein and then the E protein (“PrM/E”).
  • the protein comprises the M and E proteins (either M/E or E/M order).
  • the protein is a single chain (e.g., the mRNA encoding a single chain protein). In some embodiments, stabilizing mutations are included.
  • the E protein when the antigen is in the E/M order, is connected to the M protein via a linker (e.g., a GS linker), or a Rosetta-based linker.
  • the flavivirus fusion protein further comprises a Capsid (C) protein and the NS2b/NS3 protease sequences from the corresponding virus (e.g., DENV or ZIKV) (denoted as “C-NS2B3” in the tables below).
  • C-NS2B3 the NS2b/NS3 protease are involved in the maturation process of flaviviruses and their inclusion, it is thought, results in a higher yield of the VLPs.
  • the inclusion of a Capsid protein and NS2b/NS3 protease may result in the symmetric assembly of the E/M or M/E heterotetramers.
  • the Capsid protein is further mutated to reduce the basic charge (e.g., with an R85M mutation relative to NCBI Reference Sequence: NP_739591.2).
  • the engineered flavivirus protein comprises amino acid mutations resulting in formation of a glycosylation site.
  • the glycosylation site is introduced in an engineered ZIKV E protein, for example, using a N 67 X 68 S 69 motif relative to SEQ ID NO: 282.
  • Non-limiting examples of DENV and ZIKV and YFV proteins of the present disclosure are provided in Tables 1-4.
  • the signal peptide e.g., MLRLLLRHHFHCLLLCAVWATPCLA (SEQ ID NO: 271) or MKAILVVLLYTFTTANA (SEQ ID NO: 272)
  • SEQ ID NO: 271 MLRLLLRHHFHCLLLCAVWATPCLA
  • SEQ ID NO: 272 MLRLLLRHHFHCLLLCAVWATPCLA
  • MKAILVVLLYTFTTANA SEQ ID NO: 272
  • a flavivirus is of a particular strain of flavivirus.
  • a YFV is an Asibi strain.
  • a YFV is a 17D strain.
  • a YFV is Brazilian strain (e.g., YFV2015-2018).
  • a YFV is a Venezuelan strain (e.g., Venezuela 2000).
  • a YFV is an African strain (e.g., West Africa I, West Africa II, East Africa, East/Central Africa, or Angola).
  • YFV is a South American strain (e.g., South America I or South America II).
  • Non-limiting examples of wild-type DENV, ZIKV and YFV sequences may be found at GenBank Accession Nos: QHJ14106.1 (DENV1), QBK46950.1 (DENV2), UJT50354.1 (DENV3), UDW38823.1 (DENV4), AZS35368.1 (ZIKV), AAX47570.1/ NP_041726.1 (YFV) and are listed below.
  • a vaccine of the present disclosure comprises a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363.
  • a vaccine of the present disclosure comprises a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212- 213, 285-301, and 347-352.
  • a vaccine of the present disclosure comprises a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214.
  • a vaccine of the present disclosure comprises a nucleic acid comprising an open reading frame encoding a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246- 255, 302-346, and 353-363.
  • a vaccine of the present disclosure comprises a nucleic acid comprising an open reading frame encoding a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352.
  • a vaccine of the present disclosure comprises a nucleic acid comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214.
  • a vaccine of the present disclosure comprises a DNA comprising an open reading frame encoding a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363.
  • a vaccine of the present disclosure comprises a DNA comprising an open reading frame encoding a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352.
  • a vaccine of the present disclosure comprises a DNA comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238- 245, 187-188, and 214.
  • a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246- 255, 302-346, and 353-363.
  • a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352.
  • a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214.
  • each of the E proteins in the heterotetramer has the same amino acid substitutions as one another.
  • the two E proteins in the heterotetramer have at least one amino acid substitution different from one another.
  • each of the M proteins in the heterotetramer has the same amino acid substitution(s) as one another. In some embodiments, the two M proteins in the heterotetramer have at least one amino acid substitution different from one another.
  • the flavivirus proteins of the present disclosure are antigenic, i.e., they promote an immune response when administered to a subject.
  • the subject antigens can be administered as a protein or as a nucleic acid molecule encoding the protein. When delivered to a subject in nucleic acid form, the antigenic flavivirus heterotetramer is expressed by cells in the subject and stimulates the subject’s immune system.
  • the mRNA When delivered as an mRNA, the mRNA is provided in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles) such that upon administration to cells, tissues or subjects, the mRNA is taken up by cells which, in turn, express the protein(s) encoded by the mRNA.
  • the vaccines of the present disclosure comprise mRNA encoding the desired DENV antigen(s), which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired antigen(s).
  • the RNA is formulated (e.g., encapsulated) in a lipid nanoparticle.
  • RNA Upon delivery and uptake by cells of the body, the RNA is translated in the cytosol and the antigens are generated by the host cell machinery.
  • the antigens are presented by the host cells and elicit an adaptive humoral and cellular immune response.
  • Neutralizing antibodies are directed against the expressed antigens, and hence the antigens are, in the case of stabilized heterotetramers, relevant target antigens for use as vaccines because they closely mirror the conformation of naturally occurring flaviviruses.
  • mRNA technology is amenable to rapid design and testing of mRNA encoding a variety of antigens.
  • rapid production of mRNA coupled with formulation in appropriate delivery vehicles e.g., lipid nanoparticles
  • antigens encoded by the mRNAs of the present disclosure are expressed by the cells of the subject, e.g., are expressed by the human body, and thus the subject, e.g., the human body, serves as the “factory” to produce the antigens which, in turn, elicits the desired immune response.
  • the vaccines may include a single mRNA or multiple RNAs encoding two or more antigens of the same or different flaviviruses (e.g., different strains of a flavivirus).
  • combination vaccines that include mRNA encoding one or more DENV antigens and one or more antigen(s) of a different virus (e.g., a second flavivirus).
  • the vaccines of the present disclosure may be combination vaccines that target one or more antigens of the same strain/species, or one or more antigens of different strains/species, e.g., antigens that induce immunity to organisms that are found in the same geographic areas where the risk of flavivirus infection (e.g., DENV) is high or organisms to which an individual is likely to be exposed to when exposed to the flavivirus.
  • Engineered Flavivirus Proteins In some embodiments, the engineered flavivirus proteins comprise additional amino acid substitutions.
  • amino acid substitutions are provided below. For clarity, mutations are described using amino acid numbering corresponding to a wild-type sequence (e.g., wild-type DENV2 E protein, SEQ ID NO: 274). The person of ordinary skill in the art will appreciate that mutations disclosed in relation to the wild-type sequence may be applied to other flavivirus protein amino acid sequences. To apply a substitution disclosed with numbering corresponding to the wild-type sequence to an engineered protein, the skilled artisan may align the engineered protein’s amino acid sequence to the amino acid sequence of the wild-type sequence with which the substitution is numbered.
  • substitutions disclosed in relation to a listed amino acid sequence may be referred to in the form X1[#]X2, where X1 is the amino acid at position [#] in a listed amino acid sequence, and X2 is the amino acid introduced by replacement of the X1 at position [#].
  • a glycine substitution at position 95 may also be referred to as an A95G substitution, when using SEQ ID NO: 281 as a listed amino acid sequence, because the alanine (A) at position 95 of SEQ ID NO: 281 is replaced with a glycine (G).
  • Such a substitution may also be referred to as an “X2 substitution at position [#]”, meaning that a protein comprises an X2 residue at position [#] (numbered by alignment to a listed sequence), regardless of whether the residue that was present at position [#] in the reference amino acid sequence was X1, or a different residue other than X2.
  • DENV1 E Protein may comprise at least one amino acid substitution.
  • the DENV1 E protein comprises at least one amino acid substitution at one or more positions selected from T76, R93, G106, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to the DENV1 E protein reference sequence (SEQ ID NO: 274).
  • the DENV1 E protein comprises at least one substitution selected from T76Q, T76D, G106S, G106T, G106N, G106K, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, T268V, A280C, G445K, A495C, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises substitutions at T76D, L107R, T262S, T265A, A280C, and G445K, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises substitutions at T76Q, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76D, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and A280C, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises substitutions at T76D, G106T, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and A280C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76D, G106N, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and A280C, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, G106T, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises substitutions at T76Q, G106N, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises a T76Q substitution relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises a glutamine at position 76 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises an G106S substitution relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises a serine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises a G106T substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a threonine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a G106N substitution relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an asparagine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a G106K substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises a lysine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises an L107R substitution relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises an arginine at position 107 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises a Q131A substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 131 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an N194D substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an aspartic acid at position 194, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises an aspartic acid at position 194 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises an H209N substitution relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises an asparagine at position 209, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises an asparagine at position 209 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises a T262S substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a serine at position 262, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a serine at position 262 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a T265A substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 265, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises an alanine at position 265 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an A267C substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 267, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 267 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises a T268A substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 268, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 268 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an A280C substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 280, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises a cysteine at position 280 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises a G445K substitution relative to SEQ ID NO: 274.
  • the DENV1 E protein comprises a lysine at position 445, where the positions are numbered according to SEQ ID NO: 274.
  • the DENV1 E protein comprises a lysine at position 445 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274.
  • the DENV1 E protein comprises an A495C substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 495, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 495 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. DENV1 M Protein In some embodiments, the DENV1 M protein may comprise at least one mutation.
  • the DENV1 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, A97, H99, V100, L102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or A167, relative to the DENV1 M protein reference sequence (SEQ ID NO: 273).
  • the DENV1 M protein comprises at least one substitution selected from S93G, V94I, H99C, V100A, L104I, T106G, T106C, T108C, relative to SEQ ID NO: 273.
  • the DENV1 M protein comprises substitutions at V100A and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at S93G, V94I, H99C, V100A, L104I, T106G, and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at S93G, V94I, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at S93G, A97N, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 273.
  • the DENV1 M protein comprises an S93G substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a glycine at position 93 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a V94I substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 273.
  • the DENV1 M protein comprises an isoleucine at position 94 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273.
  • the DENV1 M protein comprises an H99C substitution relative to SEQ ID NO: 273.
  • the DENV1 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 273.
  • the DENV1 M protein comprises a cysteine at position 99 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273.
  • the DENV1 M protein comprises a V100A substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an alanine at position 100 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an L104I substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 273.
  • the DENV1 M protein comprises an isoleucine at position 104 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273.
  • the DENV1 M protein comprises a T106G substitution relative to SEQ ID NO: 273.
  • the DENV1 M protein comprises a glycine at position 106, where the positions are numbered according to SEQ ID NO: 273.
  • the DENV1 M protein comprises a glycine at position 106 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273.
  • the DENV1 M protein comprises a T108C substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a cysteine at position 108 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. DENV2 E Protein In some embodiments, the DENV2 E protein may comprise at least one mutation.
  • the DENV2 E protein comprises at least one substitution at one or more positions selected from T76, R93, G106, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to the DENV2 E protein reference sequence (SEQ ID NO: 276).
  • the DENV2 E protein comprises at least one substitution selected from T76D, T76N, T76E, T76Y, G106S, G106T, G106N, G106K, L107R, L107K, L107Q, Q131N, Q131A, Q131L, Q131E, E133A, N194E, N194D, E195Q, E195S, H209Y, H209N, H209D, H209K, T262S, T262C, T265A, A267C, T268A, T268I, T268V, T280C, H317N, T319V, G441A, G445K, A495G, A495C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76D, L107R, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and T280C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76D, L107R, T262S, T265A, H317N, T319V, G441A, and G445K, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, and A495C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262C, T265A, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, T280C, and A495C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N194E, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262C, T265A, A267C, T268V, T280C, and A495C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N194E, H209N, T262C, T265A, A267C, T268V, T280C, and A495C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N194E, H209N, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, and T280C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, A267C, T268V, and T280C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76D, G106T, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, G106N, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76D, G106K, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, G106S, L107R, Q131A, N194D, H209N, T262C, T265A, A267C, T268V, T280C, and A495C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, G106S, L107R, Q131A, L191I, N194D, H209N, T262S, T265A, A267C, T268I, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Q, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and T280C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76D substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an aspartic acid at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 76, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an asparagine at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76E substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a T76Y substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an G106S substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a G106T substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a threonine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a G106N substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an asparagine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a G106K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an L107R substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an arginine at position 107 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an L107K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 107, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a lysine at position 107 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an L107Q substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 107, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 107 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a Q131N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131A substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131L substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a leucine at position 131, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a leucine at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131E substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises an E133A substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 133, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 133 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an N194E substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises a glutamic acid at position 194, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 194 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an N194D substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 194, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an aspartic acid at position 194 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an E195Q substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 195, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 195 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises an E195S substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 195, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 195 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209Y substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises a tyrosine at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 209, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an asparagine at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209D substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises an H209K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T262S substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises a serine at position 262, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 262 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T262C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 262, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a cysteine at position 262 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a T265A substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an alanine at position 265, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an alanine at position 265 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises an A267C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 267, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 267 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T268A substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an alanine at position 268, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 268 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T268I substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an isoleucine at position 268, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an isoleucine at position 268 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a T268V substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises a valine at position 268, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a valine at position 268 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a T280C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 280, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 280 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H317N substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an asparagine at position 317, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 317 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T319V substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a valine at position 319, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a valine at position 319 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a G441A substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises an alanine at position 441, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises an alanine at position 441 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • the DENV2 E protein comprises a G445K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 445, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 445 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an A495G substitution relative to SEQ ID NO: 276.
  • the DENV2 E protein comprises a glycine at position 495, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glycine at position 495 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an A495C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 495, where the positions are numbered according to SEQ ID NO: 276.
  • the DENV2 E protein comprises a cysteine at position 495 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276.
  • DENV2 M Protein In some embodiments, the DENV2 M protein may comprise at least one mutation.
  • the DENV2 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to the DENV2 M protein reference sequence (SEQ ID NO: 275).
  • the DENV2 M protein comprises at least one substitution selected from S93G, V94I, A95C, L96F, L96I, L96W, V97N, H99C, V100A, V100C, V100I, L104H, L104I, E105L, T106G, T106A, T106S, T108C, A117Q, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at V100C, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at L96I, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96W, V97N, H99C, V100I, L104I, E105L, T106A, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96W, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96W, H99C, V100I, L104I, and T108C, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, A95C, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, T106S, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V97N, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, T106S, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V97N, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100A, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an S93G substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 93 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV2 M protein comprises a V94I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 94 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an A95C substitution relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises a cysteine at position 95, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 95 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L96F substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a phenylalanine at position 96, where the positions are numbered according to SEQ ID NO: 275.
  • the DENV2 M protein comprises a phenylalanine at position 96 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV2 M protein comprises an L96I substitution relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises an isoleucine at position 96, where the positions are numbered according to SEQ ID NO: 275.
  • the DENV2 M protein comprises an isoleucine at position 96 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L96W substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a tryptophan at position 96, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a tryptophan at position 96 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV2 M protein comprises a V97N substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an asparagine at position 97, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an asparagine at position 97 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an H99C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 275.
  • the DENV2 M protein comprises a cysteine at position 99 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V100A substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 100 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV2 M protein comprises a V100C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 100, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 100 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V100I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 100, where the positions are numbered according to SEQ ID NO: 275.
  • the DENV2 M protein comprises an isoleucine at position 100 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L104H substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a histidine at position 104, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a histidine at position 104 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV2 M protein comprises an L104I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 104 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an E105L substitution relative to SEQ ID NO: 275.
  • the DENV2 M protein comprises a leucine at position 105, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a leucine at position 105 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T106G substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 106, where the positions are numbered according to SEQ ID NO: 275.
  • the DENV2 M protein comprises a glycine at position 106 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T106A substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 106, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 106 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV2 M protein comprises a T106S substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a serine at position 106 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T108C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 275.
  • the DENV2 M protein comprises a cysteine at position 108 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an A117Q substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glutamine at position 117, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glutamine at position 117 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275.
  • the DENV3 E protein may comprise at least one mutation.
  • the DENV3 E protein comprises at least one substitution at one or more positions selected from T76, K93, G106, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A493, relative to the DENV3 E protein reference sequence (SEQ ID NO: 278).
  • the DENV3 E protein comprises at least one substitution selected from T76Q, T76K, T76E, T76Y, G106S, G106T, G106N, G106K, L107R, L107N, Q131L, Q131A, Q131N, Q131E, E133A, N192D, N192E, E193Q, H207N, H207Y, T260S, T260C, T263A, A265C, T266V, T266A, A278C, A278T, T443K, A493C, A493G, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Q, L107R, T260S, T263A, T266V, A278C, and T443K, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, and T443K, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, and T443K, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, R208K, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, R208K, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, R208K, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, L189I, N192D, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Q, G106S, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, G106T, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Q, G106N, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, T266A, A278C, T443K, and A493C, relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises substitutions at T76Q, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, T266A, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, T266A, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T76Q substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T76K substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 76, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a lysine at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T76E substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a glutamic acid at position 76, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a glutamic acid at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T76Y substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a tyrosine at position 76, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a tyrosine at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises an G106S substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a serine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a G106T substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a G106N substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a G106K substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a lysine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises an L107R substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises an arginine at position 107 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises an L107N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 107, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 107 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131L substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a leucine at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a leucine at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131A substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises an alanine at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a Q131E substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an E133A substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises an alanine at position 133, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 133 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an N192D substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an aspartic acid at position 192, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises an aspartic acid at position 192 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an N192E substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 192, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 192 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises an E193Q substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 193, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 193 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an H207N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 207, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises an asparagine at position 207 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises an H207Y substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a tyrosine at position 207, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a tyrosine at position 207 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T260S substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a serine at position 260, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a serine at position 260 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T260C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 260, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 260 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T263A substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises an alanine at position 263, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 263 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A265C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 265, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a cysteine at position 265 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T266V substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a valine at position 266, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a valine at position 266 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T266A substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 266, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 266 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A278C substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a cysteine at position 278, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 278 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A278T substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 278, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a threonine at position 278 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises a T443K substitution relative to SEQ ID NO: 278.
  • the DENV3 E protein comprises a lysine at position 443, where the positions are numbered according to SEQ ID NO: 278.
  • the DENV3 E protein comprises a lysine at position 443 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A493C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 493, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 493 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278.
  • the DENV3 E protein comprises an A493G substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glycine at position 493, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glycine at position 493 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. DENV3 M Protein In some embodiments, the DENV3 M protein may comprise at least one mutation.
  • the DENV3 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to the DENV3 M protein reference sequence (SEQ ID NO: 277).
  • the DENV3 M protein comprises at least one substitution selected from S93G, V94I, A95C, A97V, H99C, V100A, V100C, L104I, T106C, T108C, relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises substitutions at V100A and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, V100A, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100C, and L104I, relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, A97V, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100C, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, and L104I, relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, A97V, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, V100A, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100C, and T106C, relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100A, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an S93G substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a glycine at position 93 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277.
  • the DENV3 M protein comprises a V94I substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an isoleucine at position 94 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an A95C substitution relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises a cysteine at position 95, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 95 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an A97V substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a valine at position 97, where the positions are numbered according to SEQ ID NO: 277.
  • the DENV3 M protein comprises a valine at position 97 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an H99C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 99 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277.
  • the DENV3 M protein comprises a V100A substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an alanine at position 100 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a V100C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 100, where the positions are numbered according to SEQ ID NO: 277.
  • the DENV3 M protein comprises a cysteine at position 100 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277.
  • the DENV3 M protein comprises an L104I substitution relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 277.
  • the DENV3 M protein comprises an isoleucine at position 104 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277.
  • the DENV3 M protein comprises a T106C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 106, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 106 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a T108C substitution relative to SEQ ID NO: 277.
  • the DENV3 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 108 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277.
  • DENV4 E Protein In some embodiments, the DENV4 E protein may comprise at least one mutation.
  • the DENV4 E protein comprises at least one substitution at one or more positions selected from T76, R93, G106, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to the DENV4 E protein reference sequence (SEQ ID NO: 280).
  • the DENV4 E protein comprises at least one substitution selected from T76Q, T76D, T76Y, G106S, G106T, G106N, G106K, L107R, L107E, Q131A, N194E, N194R, E195Q, H209D, H209N, H209Y, A267C, T268A, A280C, T445K, A495C, or A495G, relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises substitutions at T76Q, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises substitutions at T76Q, L107R, A280C, and T445K, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106T, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises substitutions at T76Q, G106N, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, L107R, Q131A, H209D, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, H209D, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, H209D, S262C, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a T76Q substitution relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a glutamine at position 76 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an G106S substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 280.
  • the DENV4 E protein comprises a serine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a G106T substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 280.
  • the DENV4 E protein comprises a threonine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a G106N substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an asparagine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280.
  • the DENV4 E protein comprises a G106K substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a lysine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an L107R substitution relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an arginine at position 107 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a Q131A substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 280.
  • the DENV4 E protein comprises an alanine at position 131 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an H209D substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an aspartic acid at position 209, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an aspartic acid at position 209 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280.
  • the DENV4 E protein comprises an A267C substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 267, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 267 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a T268A substitution relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises an alanine at position 268, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an alanine at position 268 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an A280C substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 280, where the positions are numbered according to SEQ ID NO: 280.
  • the DENV4 E protein comprises a cysteine at position 280 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280.
  • the DENV4 E protein comprises a T445K substitution relative to SEQ ID NO: 280.
  • the DENV4 E protein comprises a lysine at position 445, where the positions are numbered according to SEQ ID NO: 280.
  • the DENV4 E protein comprises a lysine at position 445 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an A495C substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 495, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 495 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280.
  • the DENV4 M protein may comprise at least one mutation.
  • the DENV4 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, T97, H99, S100, M102, L104, E105, T106, R107, A108, E109, S113, S114, A117, K119, Q122, E125, S126, R130, S165, Y166, or G167, relative to the DENV4 M protein reference sequence (SEQ ID NO: 279).
  • the DENV4 M protein comprises at least one substitution selected from S93G, V94I, H99C, S100A, S100C, L104I, T106G, A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S100A and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, V94I, S100A, T106G, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, V94I, H99C, S100C, L104I, T106G, and A108C, relative to SEQ ID NO: 279.
  • the DENV4 M protein comprises substitutions at S93G, H99C, S100C, L104I, T106G, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, A95C, H99C, S100C, L104I, T106S, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a S93G substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 279.
  • the DENV4 M protein comprises a glycine at position 93 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279.
  • the DENV4 M protein comprises a V94I substitution relative to SEQ ID NO: 279.
  • the DENV4 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 279.
  • the DENV4 M protein comprises an isoleucine at position 94 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279.
  • the DENV4 M protein comprises an H99C substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 99 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an S100A substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 279.
  • the DENV4 M protein comprises an alanine at position 100 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an S100C substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 100, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 100 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279.
  • the DENV4 M protein comprises an L104I substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an isoleucine at position 104 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a T106G substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a glycine at position 106, where the positions are numbered according to SEQ ID NO: 279.
  • the DENV4 M protein comprises a glycine at position 106 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279.
  • the DENV4 M protein comprises an A108C substitution relative to SEQ ID NO: 279.
  • the DENV4 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 279.
  • the DENV4 M protein comprises a cysteine at position 108 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279.
  • the ZIKV E protein may comprise at least one mutation.
  • the ZIKV E protein comprises at least one substitution at one or more positions selected from D67, A69, T76, K93, L107, Q131, E133, T194, D197, S199, D200, H214, A248, T267, A270, A272, L273, S286, A319, H323, T325, R402, S403, K443, Q447, G450, A451, K454, S503, or A504, relative to the ZIKV E protein reference sequence (SEQ ID NO: 282).
  • the ZIKV E protein comprises at least one substitution selected from D67N, A69S, T76K, T76Q, T76Y, L107D, L107R, L107Q, Q131N, S199K, T267L, A270G, A272C, L273I, S286C, S403E, K454R, V494G, L495N, I496P, F497D, L498H, S499R, T500E, A501M, A501G, A501P, V502G, S503G, S503E, S503P, A504G, A504T, A504P, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at D67N and A69S relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at T76K, L107D, S286C, V494G, L495N, I496P, F497D, L498H, S499R, T500E, A501M, V502G, S503E, and A504T, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, V494G, L495N, I496P, F497D, L498H, S499R, T500E, A501M, V502G, and S503E, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at L107R, S403E, and K454R, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at L107R, S286C, S403E, and K454R, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Q, L107R, T267L, A270G, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at T76Q, L107R, T267L, A270G, A272C, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Q, L107R, T267L, A270G, A272C, S286C, A501P, V502G, S503G, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at T76Q, L107R, Q131N, T267L, A270G, A272C, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107R, Q131N, A270G, A272C, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, A272C, L273I, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, L273I, S286C, V502G, S503P, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, L273I, S286C, V502G, S503G, and A504P, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, A272C, L273I, S286C, V502G, S503G, and A504P, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at T76Y, L107Q, Q131N, S199K, A272C, L273I, S286C, V502G, S503G, and A504P, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, G106S, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, G106T, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at , relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, G106N, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at D67N, A69S, T76K, G106S, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises substitutions at D67N, A69S, T76K, G106T, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at D67N, A69S, T76K, G106N, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a D67N substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 67, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an asparagine at position 67 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A69S substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a serine at position 69, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a serine at position 69 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises a T76K substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 76, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 76 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T76Q substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 76 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T76Y substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a tyrosine at position 76, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a tyrosine at position 76 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an L107D substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises an aspartic acid at position 107, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an aspartic acid at position 107 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an L107R substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an arginine at position 107 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L107Q substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 107, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 107 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises a Q131N substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 131, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 131 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S199K substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a lysine at position 199, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 199 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T267L substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a leucine at position 267, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a leucine at position 267 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A270G substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 270, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 270 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A272C substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a cysteine at position 272, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a cysteine at position 272 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an L273I substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises an isoleucine at position 273, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an isoleucine at position 273 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an S286C substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a cysteine at position 286, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a cysteine at position 286 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an S403E substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glutamic acid at position 403, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glutamic acid at position 403 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises a K454R substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises an arginine at position 454, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an arginine at position 454 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises a V494G substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 494, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 494 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an L495N substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises an asparagine at position 495, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an asparagine at position 495 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an I496P substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 496, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 496 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an F497D substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an aspartic acid at position 497, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an aspartic acid at position 497 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L498H substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a histidine at position 498, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a histidine at position 498 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S499R substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 499, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises an arginine at position 499 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises a T500E substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glutamic acid at position 500, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glutamic acid at position 500 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A501M substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a methionine at position 501, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a methionine at position 501 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A501G substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 501, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 501 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A501P substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 501, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 501 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises a V502G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 502, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 502 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S503G substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glycine at position 503, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 503 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S503E substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 503, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a glutamic acid at position 503 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an S503P substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 503, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 503 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • the ZIKV E protein comprises an A504G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 504, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 504 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A504T substitution relative to SEQ ID NO: 282.
  • the ZIKV E protein comprises a threonine at position 504, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a threonine at position 504 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A504P substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 504, where the positions are numbered according to SEQ ID NO: 282.
  • the ZIKV E protein comprises a proline at position 504 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282.
  • ZIKV M Protein In some embodiments, the ZIKV M protein may comprise at least one mutation.
  • the ZIKV M protein comprises at least one substitution at one or more positions selected from A95, V96, T97, L98, P99, H101, S102, R104, L106, Q107, T108, R109, S110, Q111, E115, S116, Y119, K121, I124, E127, N128, R132, A167, Y168, or S169, relative to the ZIKV M protein reference sequence (SEQ ID NO: 281).
  • the ZIKV M protein comprises at least one substitution selected from A95G, A95S, V96I, V96L, T97F, H101C, S102A, T108S, S110C, E115S, Y119H, A167C, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, S102A, S110C, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95S, V96I, S110C, and Y119H, relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises substitutions at S102A, S110C, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, S110C, E115S, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, S110C, E115S, Y119H, and A167C, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, H101C, S110C, E115S, and Y119H, relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises substitutions at V96L, T97F, H101C, S110C, E115S, Y119H, and A167C, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, H101C, S102A, T108S, S110C, E115S, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, S102A, T108S, S110C, E115S, and Y119H, relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises substitutions at A95G, V96I, H101C, S102A, T108S, S110C, E115S, and Y119H, relative to SEQ ID NO: 281.. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, S102A, S110C, E118D, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an A95G substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a glycine at position 95, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises a glycine at position 95 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises an A95S substitution relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises a serine at position 95, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises a serine at position 95 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises a V96I substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an isoleucine at position 96, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an isoleucine at position 96 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a V96L substitution relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises a leucine at position 96, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a leucine at position 96 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a T97F substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a phenylalanine at position 97, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises a phenylalanine at position 97 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises an H101C substitution relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises a cysteine at position 101, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises a cysteine at position 101 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises an S102A substitution relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises an alanine at position 102, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises an alanine at position 102 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises a T108S substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 108, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 108 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an S110C substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 110, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises a cysteine at position 110 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises an E115S substitution relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises a serine at position 115, where the positions are numbered according to SEQ ID NO: 281.
  • the ZIKV M protein comprises a serine at position 115 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281.
  • the ZIKV M protein comprises a Y119H substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a histidine at position 119, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a histidine at position 119 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an A167C substitution relative to SEQ ID NO: 281.
  • the ZIKV M protein comprises a cysteine at position 167, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 167 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. Flavivirus Proteins – Additional Amino Acid Substitution Positions In some embodiments, the flavivirus E protein may comprise at least one mutation.
  • the flavivirus E protein comprises at least one substitution at one or more positions selected from (i) T76, R93, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to SEQ ID NOs: 274, 276 or 280; or (ii) T76, K93, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A493, relative to SEQ ID NO: 278; or (iii) T76, K93, L107, Q131, E133, T189, D192
  • the flavivirus M protein may comprise at least one mutation.
  • the flavivirus M protein comprises at least one substitution at one or more positions selected from (i) S93, V94, A95, L96, A97, H99, V100, L102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or A167, relative to SEQ ID NO: 273; or (ii) S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to SEQ ID NO: 275 or 277; or (iii) S93, V94, A95, L96, T97, H99, S100, M102, L
  • compositions of the present disclosure include mRNA that encodes a flavivirus protein comprising an amino acid substitution at positions 107 and 76 corresponding to DENV2 E protein (e.g., SEQ ID NO: 276), and may comprise at least one additional amino acid substitution.
  • Such proteins may possess one or more substitutions, deletions, and/or insertions at certain positions within its amino acid sequence, as compared to a naturally occurring or reference amino acid sequence.
  • a protein variant has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to a naturally occurring or reference sequence, and maintain the stabilized heterodimer formation that induces optimal immune responses.
  • Global sequence alignment and local sequence alignment are two common methods used to compare and analyze sequences of DNA, RNA, or protein. Global sequence alignment compares the entire length of two sequences and finds the best possible alignment of the entire length of the sequences. It is useful, for example, when the two sequences being compared are similar in length and share significant homology. Local sequence alignment, on the other hand, identifies regions of similarity between sequences, allowing for gaps and mismatches in the alignment. This method is useful for identifying short regions of homology within larger sequences, and can be used to identify functional domains, protein families, and binding sites. Local alignment can be computationally more efficient than global alignment, and can be applied to sequences of different lengths.
  • percent (%) identity between two mRNA polynucleotides or between two proteins refers to percent (%) identity determined using a global sequence alignment, comparing the length of two comparable sequences (e.g., E protein to a second E protein, M protein to a second M protein, mRNA encoding an E protein to mRNA encoding a second E protein (ORF, or entire mRNA), or mRNA encoding an M protein to mRNA encoding a second M protein (ORF or entire mRNA).
  • Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • variants of a particular polynucleotide or polypeptide have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular naturally occurring or reference sequence as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • tools for alignment include but are not limited to those of the BLAST suite (Altschul, S.F., et al. Nucleic Acids Res.1997;25:3389-3402); and those based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. J. Mol. Biol.1981;147:195- 197).
  • a general global alignment technique based on dynamic programming is the Needleman– Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. J. Mol. Biol.1920;48:443-453).
  • a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) also has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman–Wunsch algorithm.
  • Needle from EMBOSS
  • Clustal Omega Clustal Omega
  • MUSCLE Multiple Sequence Comparison by Log-Expectation
  • MAFFT Multiple Alignment using Fast Fourier Transform
  • T-Coffee Tree-based Consistency Objective Function for Alignment Evaluation
  • an mRNA comprises an open reading frame that encodes a DENV protein comprising the amino acid sequence of any one of the sequences provided herein or comprising an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence of any one of the sequences provided herein.
  • an mRNA comprises an open reading frame that encodes a ZIKV protein comprising the amino acid sequence of any one of the sequences provided herein or comprising an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence of any one of the sequences provided herein. See, e.g., SEQ ID NOs: 22- 25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352.
  • a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises the amino acid sequence of any one of the sequences provided herein or comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238- 245, 187-188, and 214.
  • polypeptide sequences containing substitutions, insertions and/or deletions (e.g., indels), and covalent modifications with respect to naturally occurring or reference sequence, for example, the polypeptide (e.g., protein) sequences disclosed herein, are included within the scope of this disclosure.
  • sequence tags or amino acids such as one or more lysine(s) can be added to polypeptide sequences (e.g., at the N-terminal and/or C-terminal end). Sequence tags can be used for peptide detection, purification and/or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • amino acid residues located at the N-terminal and/or C-terminal regions of the amino acid sequence of a protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminal or N-terminal amino acids
  • sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., foldon regions) and the like are substituted with alternative sequences that achieve the same or a similar function.
  • cavities in the core of proteins can be filled to further improve stability, e.g., by introducing larger amino acids.
  • buried hydrogen bond networks are replaced with hydrophobic resides to improve stability.
  • glycosylation sites are removed and replaced with appropriate residues.
  • sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an mRNA vaccine.
  • protein variants of stabilized heterotetramers comprising substitutions at amino acid positions 107 and 76 corresponding to a DENV2 E reference sequence will maintain the stabilized heterotetramer confirmation such that an appropriate immune response can be generated.
  • Non-limiting examples of protein variants are provided in Tables 1-4.
  • Signal Peptides In some embodiments, an mRNA has an open reading frame that encodes a signal peptide fused to the flavivirus protein. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway.
  • the signal peptide of a nascent precursor protein directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing.
  • ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor.
  • a signal peptide may also facilitate the targeting of the protein to the cell membrane.
  • a signal peptide may have a length of 15-60 amino acids.
  • a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.
  • a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15- 20 amino acids.
  • a signal peptide used in the present disclosure comprises MLRLLLRHHFHCLLLCAVWATPCLA (SEQ ID NO: 271).
  • a signal peptide used in the present disclosure comprises MKAILVVLLYTFTTANA (SEQ ID NO: 272). Fusion Proteins Some aspects of the present disclosure provide fusion proteins.
  • an mRNA encodes a fusion protein (e.g., a fusion protein comprising a flavivirus E protein and a flavivirus M protein).
  • an encoded protein may include two or more proteins (e.g., protein and/or protein fragment) joined together with or without a linker. Fusion proteins, in some embodiments, retain the functional property of each independent (nonfusion) protein.
  • a fusion protein comprises 2, 3, 4, 5, 6, or 7 of the following flavivirus proteins: prM protein, M protein, C protein, and E protein.
  • a fusion protein comprises 2, 3, 4, 5, 6, or 7 of the following flavivirus proteins: prM protein, M protein, E protein, and C protein.
  • a fusion protein comprises 2, 3, 4, 5, 6, or 7 of the following flavivirus proteins: prM protein, M protein, E protein, C protein, NS1 protein, NS2A protein, NS2B protein, NS4A protein, NS4B protein, NS3 protein, and NS5 protein.
  • an mRNA that encodes a flavivirus protein of the disclosure further encodes a linker located between at least one or each protein of the fusion protein.
  • the linker is a GS linker.
  • GS linkers are polypeptide linkers that include glycine and serine amino acids repeats.
  • an mRNA encodes a fusion protein that comprises a GS linker that is 3 to 20 amino acids long.
  • the GS linker may have a length of (or have a length of at least) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • a GS linker is (or is at least) 15 amino acids long (e.g., GGSGGSGGSGGSGGG (SEQ ID NO: 155)).
  • a GS linker is (or is at least) 8 amino acids long (e.g., GGGSGGGS (SEQ ID NO: 149)). In some embodiments, a GS linker is (or is at least) 7 amino acids long (e.g., GGGSGGG (SEQ ID NO: 150)). In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GGGS (SEQ ID NO: 151)). In some embodiments, the GS linker comprises (GGGS)n (SEQ ID NO: 152), where n is any integer from 1-5.
  • a GS linker is (or is at least) 4 amino acid long (e.g., GSGG (SEQ ID NO: 153)).
  • the GS linker comprises (GSGG)n (SEQ ID NO: 154), where n is any integer from 1-5.
  • a linker is a glycine linker, for example having a length of (or a length of at least) 3 amino acids (e.g., GGG).
  • the linker comprises GGGG (SEQ ID NO: 364).
  • the linker comprises GGPG (SEQ ID NO: 365).
  • a protein encoded by an mRNA includes two or more linkers, which may be the same or different from each other.
  • the linker may be, for example, a cleavable linker or protease-sensitive linker.
  • the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof (see, e.g., WO 2017/127750).
  • This family of self- cleaving peptide linkers, referred to as 2A peptides has been described in the art (see, e.g., Kim, J.H. et al. PLoS ONE 2011;6:e18556).
  • the linker is an F2A linker.
  • the skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure).
  • Stabilization Domains are protein sequences or structures that can enhance the stability of a protein to various environmental stresses, such as temperature, pH, and proteolysis.
  • Non-limiting examples of protein stabilization domains for use to stabilize a flavivirus protein expressed by an mRNA include: lumazine synthase, ferritin, and thioredoxin.
  • a flavivirus protein is fused to a lumazine synthase.
  • Lumazine synthase is protein from bacteria and plants that can stabilize fusion partners by forming homodimers or oligomers, which can enhance the solubility and stability of the target protein.
  • a flavivirusprotein is fused to ferritin.
  • Ferritin is a protein found in animals, plants, and bacteria that can form a cage-like structure that can store and sequester iron ions, protecting the cell from oxidative damage. Fusion of a target protein with ferritin can improve its stability and solubility.
  • a flavivirus protein e.g., an E protein, is fused to thioredoxin.
  • any one of the engineered flavivirus proteins or flavivirus fusion proteins may be encoded by nucleic acids.
  • Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides.
  • Nucleic acids may be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ - L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimeras and/or combinations thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • TAA glycol nucleic acid
  • PNA peptide nucleic acid
  • LNA locked nu
  • mRNA of the present disclosure comprises an open reading frame (ORF) encoding at least one flavivirus protein.
  • the mRNA further comprises a 5 ⁇ untranslated region (UTR), 3 ⁇ UTR, a poly(A) tail and/or a 5 ⁇ cap analog.
  • UTR 5 ⁇ untranslated region
  • Preferred mRNA molecules are chemically modified.
  • Messenger RNA Messenger RNA (mRNA) is RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo.
  • mRNA is not self-amplifying RNA (saRNA) (see, e.g., Bloom K et al. Gene Therapy 2021; 28: 117–129 for a comparison of mRNA and saRNA).
  • saRNAs include alphavirus replicase sequences that encode an RNA-dependent RNA polymerase. mRNA does not include alphavirus replicase sequences.
  • nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s.
  • any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.”
  • Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to, UTRs at their 5′-end (5′ UTR) and/or at their 3′-end (3′ UTR), in addition to other structural features, such as a 5′-cap structure or a 3′-poly(A) tail. Both the 5′ UTR and the 3′ UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA.
  • UTRs Untranslated Regions
  • the mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region.
  • a “5′ untranslated region” refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
  • a “3′ untranslated region” 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.
  • the 5’ UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.
  • the mRNA may comprise a 5’ UTR and/or 3’ UTR. UTRs of an mRNA are transcribed but not translated.
  • the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule.
  • the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • a variety of 5’ UTR and 3’ UTR sequences are known.
  • the mRNA of the present disclosure may include any 5’ UTR and/or any 3’ UTR.
  • Exemplary UTR sequences include SEQ ID NOs: 256-270; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein.
  • a 5' UTR of the present disclosure comprises a sequence selected from: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 256), GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC (SEQ ID NO: 257), GAGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAA CUAGCAAGCUUUUGUUCUCGCC (SEQ ID NO: 258), and GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUAGUUUUCUCGCAACU AGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 259).
  • a 3' UTR of the present disclosure comprises a sequence selected from UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 260), UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 261), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGACUAGUGA AUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 262), UAAAGCUCCCCGGGGGCCUC
  • a 3′ UTR comprises, in 5′-to-3′ order: (a) the nucleic acid sequence UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAG (SEQ ID NO: 269), (b) an identification and ratio determination (IDR) sequence, and (c) the nucleic acid sequence UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 270). IDR sequences are described herein in the section entitled “Identification and Ratio Determination (IDR) Sequences.” UTRs may also be omitted from the mRNA provided herein.
  • a 5 ⁇ UTR does not encode a protein (is non-coding).
  • Natural 5′ UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5′UTR also have been known to form secondary structures which are involved in elongation factor binding.
  • a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF.
  • a 5’ UTR is a synthetic UTR, i.e., does not occur in nature.
  • Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic.
  • Exemplary 5’ UTRs include Xenopus or human derived a-globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, US9012219).
  • CMV immediate-early 1 (IE1) gene (US2014/0206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 232) (WO2014/144196) may also be used.
  • a 5' UTR is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO2015/101414, WO2015/101415, WO2015/062738, WO2015/024667, WO2015/024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO/2015101414, WO2015101415, WO/2015/062738), 5' UTR element derived from the 5' UTR of an hydroxysteroid (17- ⁇ ) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015/024667) can be used.
  • L32 ribosomal protein Large 32
  • HSD17B4 hydroxysteroid
  • HSD17B4 hydroxysteroid
  • WO2015024667 or a 5' UTR element derived from
  • an internal ribosome entry site is used instead of a 5' UTR.
  • a 3 ⁇ UTR does not encode a protein (is non-coding).
  • Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs.
  • AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes.
  • AREs 3′ UTR AU rich elements
  • AREs 3′ UTR AU rich elements
  • one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection.
  • cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 1 day, 2 days, and 7 days post-transfection.
  • 5’ UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence.
  • a heterologous or synthetic 5’ UTR may be used with a synthetic 3’ UTR or with a heterologous 3’ UTR.
  • Non-UTR sequences may also be used as regions or subregions within a nucleic acid.
  • introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure.
  • the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • 5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US2010/0293625 and WO2015/085318A2, each of which is herein incorporated by reference.
  • any UTR from any gene may be incorporated into the regions of a nucleic acid.
  • multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3′ UTR or 5′ UTR may be altered relative to a wild-type/native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.
  • a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used.
  • a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
  • a double beta-globin 3′ UTR may be used as described in US2010/0129877, which is incorporated herein by reference. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property.
  • polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
  • the untranslated region may also include translation enhancer elements (TEE).
  • TEE translation enhancer elements
  • the TEE may include those described in US 2009/0226470, herein incorporated by reference, and those known in the art.
  • Open Reading Frames An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA).
  • An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5′ and/or 3′ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an mRNA of the present disclosure.
  • an mRNA comprises a 5′ terminal cap.
  • 5′-capping of polynucleotides may be completed concomitantly during an in vitro transcription reaction using, for example, 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 mRNA may be completed post-transcriptionally using, for example, a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich
  • Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase 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′-O 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′-O methyl-transferase.
  • Enzymes may be derived from a recombinant source. Other cap analogs may be used.
  • poly(A) tail is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates.
  • a poly(A) tail may contain 10 to 300 adenosine monophosphates. It can, in some instances, comprise up to about 400 adenine nucleotides.
  • a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
  • a poly(A) tail contains 50 to 250 adenosine monophosphates.
  • the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.
  • the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.
  • a poly(A) tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides.
  • a poly(A) tail has a length of 100 nucleotides.
  • Stabilizing elements may include, for example, a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified.
  • SLBP stem-loop binding protein
  • SLBP RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop.
  • the minimum binding site includes at least three nucleotides 5’ and two nucleotides 3′ relative to the stem-loop.
  • an mRNA includes an open reading frame (coding region), a histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal.
  • the poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein.
  • the encoded protein in some embodiments, is not a histone protein, a reporter protein (e.g., Luciferase, GFP, EGFP, ⁇ -Galactosidase, EGFP), or a marker or selection protein (e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).
  • a reporter protein e.g., Luciferase, GFP, EGFP, ⁇ -Galactosidase, EGFP
  • a marker or selection protein e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)
  • GPT galanine phosphoribosyl transferase
  • an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop
  • an mRNA does not include a histone downstream element (HDE).
  • Histone downstream element includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3′ of naturally-occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.
  • the nucleic acid does not include an intron.
  • an mRNA may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.
  • the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure.
  • the unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region.
  • wobble base pairing (non-Watson-Crick base pairing) may result.
  • the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.
  • an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3 ’UTR. The AURES may be removed from the mRNA. Alternatively, the AURES may remain in the mRNA. Sequence Optimization In some embodiments, an open reading frame encoding a protein of the disclosure is codon optimized. Codon optimization methods are known in the art. An open reading frame of any one or more of the sequences provided herein may be codon optimized.
  • Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • encoded protein e.g., glycosylation sites
  • add, remove or shuffle protein domains add or delete restriction sites
  • modify ribosome binding sites and mRNA degradation sites adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization tools, algorithms and services are known in the art – non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
  • the open reading frame sequence is optimized using optimization algorithms.
  • Chemically Unmodified Nucleotides In some embodiments, an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT).
  • Chemically Modified Nucleotides comprise, in some embodiments, a nucleic acid molecule encoding a flavivirus stabilized heterotetramer protein, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art.
  • nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides.
  • modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally-occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
  • a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally-occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
  • a non-naturally-occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art.
  • Non-limiting examples of such non-naturally-occurring modified nucleotides and nucleosides can be found, inter alia, in international publication numbers WO2013052523A1; WO2014093924A1; WO2015051173A2; WO2015051169A2; WO2015089511A2; or WO2017153936A1, each of which is herein incorporated by reference in its entirety.
  • nucleic acids of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
  • Nucleic acids of the disclosure e.g., DNA and RNA, such as mRNA
  • nucleic acids of the disclosure in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides.
  • a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
  • a modified mRNA introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • a modified mRNA introduced into a cell or organism may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • Nucleic acids e.g., RNA, such as mRNA
  • the modifications may be present on internucleotide 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 nucleic acid may be chemically modified.
  • the present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA, such as mRNA).
  • 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”).
  • organic base e.g., a purine or pyrimidine
  • nucleobase also referred to herein as “nucleobase”.
  • 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 non-natural nucleosides.
  • Nucleic acids can comprise a region or regions of linked nucleosides.
  • Such regions may have variable backbone linkages.
  • the linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
  • 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 bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification.
  • modified nucleobases in nucleic acids comprise 1-methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy- uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine ( ⁇ ).
  • modified nucleobases in nucleic acids comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5- methoxy cytidine.
  • the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.
  • modified nucleobases in nucleic acids comprise N1-methyl-pseudouridine (m1 ⁇ ), N1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy- uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and/or pseudouridine ( ⁇ ).
  • modified nucleobases in nucleic acids comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5- methyl cytidine, and/or 5-methoxy cytidine.
  • the RNA includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.
  • a mRNA comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the mRNA.
  • the ORF comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the ORF.
  • the mRNA comprises nucleosides consisting of m1 ⁇ , adenosine, guanosine, and cytidine.
  • the ORF comprises nucleosides consisting of m1 ⁇ , adenosine, guanosine, and cytidine.
  • a mRNA comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the mRNA.
  • the ORF comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF.
  • the mRNA comprises nucleosides consisting of m1 ⁇ , adenosine, guanosine, and 5-methyl cytidine.
  • the ORF comprises nucleosides consisting of m1 ⁇ , adenosine, guanosine, and 5-methyl cytidine.
  • a mRNA comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the mRNA.
  • the ORF comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the ORF.
  • the mRNA comprises nucleosides consisting of ⁇ , adenosine, guanosine, and cytidine.
  • the ORF comprises nucleosides consisting of ⁇ , adenosine, guanosine, and cytidine.
  • a mRNA comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all cytidine positions of the mRNA.
  • the ORF comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF.
  • the mRNA comprises nucleosides consisting of ⁇ , adenosine, guanosine, and 5-methyl cytidine.
  • the ORF comprises nucleosides consisting of ⁇ , adenosine, guanosine, and 5-methyl cytidine.
  • a mRNA comprises uridine at one or more or all uridine positions of the mRNA.
  • the ORF comprises uridine at one or more or all uridine positions of the ORF.
  • the mRNA comprises nucleosides consisting of uridine, adenosine, guanosine, and cytidine.
  • the ORF comprises nucleosides consisting of uridine, adenosine, guanosine, and cytidine.
  • a mRNA comprises 5-methyl-uridine and 5-methyl cytidine at one or more or all uridine and cytidine positions, respectively, of the mRNA.
  • the ORF comprises 5-methyl-uridine substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF.
  • the mRNA comprises nucleosides consisting of 5-methyl- uridine, adenosine, guanosine, and 5-methyl cytidine.
  • the ORF comprises nucleosides consisting of 5-methyl-uridine, adenosine, guanosine, and 5-methyl cytidine.
  • a mRNA comprises 5-methyl-uridine and 5-methyl cytidine at one or more or all uridine and cytidine positions, respectively, of the mRNA.
  • RNAs e.g., mRNAs
  • RNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine.
  • nucleic acid 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.
  • the nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • 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
  • nucleotides X in a nucleic acid of the present disclosure are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • the nucleic acid 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% to 80%, 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
  • the mRNA 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.
  • the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine.
  • 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 nucleic acid 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).
  • cytosine in the nucleic acid 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).
  • an Identification and Ratio Determination (IDR) sequence is a sequence of a biological molecule (e.g., nucleic acid or protein) that, when combined with the sequence of a target biological molecule, serves to identify the target biological molecule.
  • an IDR sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and can be used as a reference to identify the target molecule.
  • a nucleic acid e.g., mRNA
  • a target sequence of interest e.g., a coding sequence encoding an antigenic peptide or protein
  • a unique IDR sequence e.g., a unique IDR sequence.
  • RNA species may comprise an IDR sequence that differs from the IDR sequence of other RNA species (e.g., RNA(s) having different coding sequence(s)).
  • Each IDR sequence thus identifies a particular RNA species, and so the abundance of IDR sequences may be measured to determine the abundance of each RNA species in a composition.
  • Use of distinct IDR sequences to identify RNA species allows for analysis of multivalent RNA compositions (e.g., containing multiple RNA species) containing RNA species with similar coding sequences and/or lengths, which could otherwise be difficult to distinguish using PCR- or chromatography-based analysis of full-length RNAs.
  • Each RNA species in a multivalent RNA composition may comprise an IDR sequence that is not a sequence isomer of an IDR sequence of another RNA species in a multivalent RNA composition (e.g., the IDR sequence does not have the same number of adenosine nucleotides, the same number of cytosine nucleotides, the same number of guanine nucleotides, and the same number of uracil nucleotides, as another IDR sequence in the composition, even if those sequences have different sequences).
  • Having identical nucleotide compositions causes sequence isomers to have the same mass, presenting a challenge to distinguishing sequence isomers using mass-based identification methods (e.g., mass spectrometry).
  • Each RNA species in a multivalent RNA composition may comprise an IDR sequence having a mass that differs from the mass of IDR sequences of each other RNA species in a multivalent RNA composition.
  • the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da.
  • Use of IDR sequences with distinct masses allows RNA fragments comprising different IDR sequences to be distinguished using mass-based analysis methods (e.g., mass spectrometry), which do not require reverse transcription, amplification, or sequencing of RNAs.
  • Each RNA species in an RNA composition may comprises an IDR sequence with a different length.
  • each IDR sequence may have a length independently selected from 0 to 25 nucleotides.
  • the length of a nucleic acid influences the rate at which the nucleic acid traverses a chromatography column, and so the use of IDR sequences of different lengths on different RNA species allows RNA fragments having different IDR sequences to be distinguished using chromatography-based methods (e.g., LC-UV).
  • IDR sequences may be chosen such that no IDR sequence comprises a start codon, ‘AUG’. Lack of a start codon in an IDR sequence prevents undesired translation of nucleotide sequences within and/or downstream from the IDR sequence.
  • IDR sequences may be chosen such that no IDR sequence comprises a recognition site for a restriction enzyme.
  • no IDR sequence comprises a recognition site for XbaI, ‘UCUAG’.
  • Lack of a recognition site for a restriction enzyme e.g., XbaI recognition site ‘UCUAG’) allows the restriction enzyme to be used in generating and modifying a DNA template for in vitro transcription, without affecting the IDR sequence or sequence of the transcribed RNA or DNA plasmid.
  • Nucleic Acid Production Chemical Synthesis Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution.
  • Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.
  • the synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.
  • the synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure.
  • the use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation Assembling nucleic acids by a ligase may also be used.
  • DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond.
  • Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.
  • RNA of the present disclosure is prepared in accordance with any one or more of the methods described in WO 2018/053209 or WO 2019/036682, each of which is incorporated by reference herein.
  • the mRNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript.
  • the template DNA is isolated DNA.
  • the template DNA is cDNA.
  • the cDNA is formed by reverse transcription of an RNA, for example, but not limited to DENV mRNA.
  • cells e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template.
  • the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified.
  • the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest.
  • plasmid DNA refers to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently.
  • plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation).
  • plasmid DNA comprises an origin of replication, which may contain one or more heterologous nucleic acids, for example nucleic acids encoding therapeutic proteins that may serve as a template for RNA polymerase.
  • Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by a restriction enzyme digest).
  • an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a poly(A) tail.
  • the particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.
  • a nucleic acid e.g., template DNA and/or RNA
  • a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.
  • An in vitro transcription system typically comprises a transcription buffer (e.g., with magnesium), nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase (e.g., T7 RNA polymerase).
  • NTPs nucleotide triphosphates
  • RNase inhibitor e.g., T7 RNA polymerase
  • a polymerase e.g., T7 RNA polymerase
  • one or more of the NTPs is a chemically modified NTP (e.g., with 1-methylpseudouridine or other chemical modifications described herein and/or known in the art).
  • the NTPs comprise adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), or an analog of each respective NTP.
  • the ratio of NTPs may vary. In some embodiments, the ratio of GTP:ATP:CTP:UTP is 1:1:1:1. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof. In some embodiments, the amount of the GTP is greater than the amount of the UTP. In some embodiments, the amount of ATP is greater than the amount of UTP, and the amount of CTP is greater than the amount of UTP.
  • an IVT system comprises an at least 2:1 ratio of GTP concentration to ATP concentration, an at least 2:1 ratio of GTP concentration to CTP concentration, and an at least 4:1 ratio of GTP concentration to UTP concentration.
  • an IVT system comprises a 2:1 ratio of GTP concentration to ATP concentration, a 2:1 ratio of GTP concentration to CTP concentration, and a 4:1 ratio of GTP concentration to UTP concentration.
  • an IVT system comprises guanosine diphosphate (GDP).
  • an IVT system comprises an at least 3:1 ratio of GTP plus GDP concentration to ATP concentration, an at least 6:1 ratio of GTP plus GDP concentration to CTP concentration, and an at least 6:1 ratio of GTP plus GDP concentration to UTP concentration.
  • the NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
  • the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. Any number of RNA polymerases or variants may be used in the method of the present disclosure.
  • the polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase.
  • An IVT system in some embodiments, comprises magnesium buffer, dithiothreitol (DTT) spermidine, pyrophosphatase, and/or RNase inhibitor. In some embodiments, an IVT system omits an RNase inhibitor.
  • An IVT system may be incubated at 25 degrees Celsius or at 37 degrees Celsius. Other temperatures may be used, depending in part on the polymerase (e.g., use of a variant polymerase).
  • the RNA transcript is capped via enzymatic capping.
  • the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp. Purification Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control.
  • Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • purification methods such as, but not limited to, strong anion exchange HPLC
  • a purified nucleic acid e.g., DNA and RNA
  • a purified nucleic acid is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.
  • a quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
  • the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. Protein Synthesis Any one of the proteins described herein may be synthesized using methods known in the art.
  • the proteins may be synthesized in vitro using a cell-free protein synthesis system (e.g., in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis).
  • a cell-free protein synthesis system e.g., in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis.
  • the proteins are purified (e.g., using affinity, ion exchange, hydrophobic interaction, and/or size exclusion) and then administered using techniques known in the art.
  • Lipid Compositions the nucleic acids are in (e.g., formulated as) a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and/or a lipoplex.
  • nucleic acids of the present disclosure are in (e.g., formulated as) lipid nanoparticle (LNP) compositions.
  • LNP lipid nanoparticle
  • Lipid nanoparticles typically comprise amino lipid, non- cationic lipid, structural lipid, and PEG lipid components along with the nucleic acid cargo (e.g., RNA, such as mRNA) of interest.
  • a lipid nanoparticles of the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575; PCT/US2016/069491; PCT/US2016/069493; and PCT/US2014/066242, all of which are incorporated by reference herein in their entirety.
  • a lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)- modified lipid.
  • a lipid nanoparticle comprises 20-60 mole percent (mol%) ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.
  • a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-30 mol% non-cationic lipid, 10-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.
  • a lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.
  • a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid.
  • a lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid.
  • a lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid.
  • a lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 45-55 mol% ionizable amino lipid.
  • lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.
  • R 1 is NR N -C4-10 cycloalkenyl optionally substituted with one or more oxo or -N(R
  • the ionizable lipid is of Formula (IL*-I): (IL*-Ia) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; and R 3a is C1-8 alkyl.
  • ionizable lipid is of Formula (IL*-Ia): or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for Formula IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-Ia’): or a salt thereof, wherein: o, M, M’, R 2c and R 3c are as defined for variable IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIa): (IL*-IIa) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for Formula IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-II’): (IL*-II’) or a salt thereof, wherein: o, M, M’, R 2c and R 3c are as defined for variable IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-III): (IL*-III) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2b is a C1-8 alkyl; and R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C 1-8 alkyl; and R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa’): (IL*-IIIa’) or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIb): (IL*-IIIb) or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIb’): (IL*-IIIb’) or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IV): (IL*-IV) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2b is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IVa): (IL*-IVa) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2b is a C1-8 alkyl; and R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-Iva’): (IL*-IVa) or a salt thereof, wherein: o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • Variables o, R 1 , R N , R N’ , R N’’ of Ionizable Lipid In some embodiments of the ionizable lipid, o is 1. In some embodiments of the ionizable lipid, o is 2. In some embodiments of the ionizable lipid, o is 3.
  • o is 4. In some embodiments of the ionizable lipid, R 1 is -OH. In some embodiments of the ionizable lipid, R N is H. In some embodiments of the ionizable lipid, R N is methyl. In some embodiments of the ionizable lipid, R N is ethyl. In some embodiments of the ionizable lipid, R 1 is -NR N -cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(R N’ R N’’ ). In some embodiments of the ionizable lipid, R N’ is H.
  • R N’ is methyl. In some embodiments of the ionizable lipid, R N’ is ethyl. In some embodiments of the ionizable lipid, R N’’ is H. In some embodiments of the ionizable lipid, R N’’ is methyl. In some embodiments of the ionizable lipid, R N’’ is ethyl. In some embodiments of the ionizable lipid, R N’ is H and R N’’ is methyl.
  • m is 4. In some embodiments of the ionizable lipid, m is 5. In some embodiments of the ionizable lipid, m is 6. In some embodiments of the ionizable lipid, m is 7. In some embodiments of the ionizable lipid, m is 8. In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, n is 5. In some embodiments of the ionizable lipid, n is 6.
  • R 2a is hydrogen. In some embodiments of the ionizable lipid, R 2a is methyl. In some embodiments of the ionizable lipid, R 2a is ethyl. In some embodiments of the ionizable lipid, R 2a is propyl. In some embodiments of the ionizable lipid, R 2a is butyl. In some embodiments of the ionizable lipid, R 2a is pentyl. In some embodiments of the ionizable lipid, R 2a is hexyl. In some embodiments of the ionizable lipid, R 2a is heptyl.
  • R 2a is octyl.
  • R 2b is hydrogen.
  • R 2b is methyl.
  • R 2b is ethyl.
  • R 2b is propyl.
  • R 2b is butyl.
  • R 2b is pentyl.
  • R 2b is hexyl.
  • R 2b is heptyl. In some embodiments of the ionizable lipid, R 2b is octyl. In some embodiments of the ionizable lipid, R 2a is hydrogen and R 2b is hydrogen. In some embodiments of the ionizable lipid, R 2a is hexyl and R 2b is hydrogen. In some embodiments of the ionizable lipid, R 2a is octyl and R 2b is hydrogen. In some embodiments of the ionizable lipid, R 2a is hydrogen and R 2b is butyl. In some embodiments of the ionizable lipid, R 2c is methyl.
  • R 2c is ethyl. In some embodiments of the ionizable lipid, R 2c is propyl. In some embodiments of the ionizable lipid, R 2c is butyl. In some embodiments of the ionizable lipid, R 2c is pentyl. In some embodiments of the ionizable lipid, R 2c is hexyl. In some embodiments of the ionizable lipid, R 2c is heptyl. In some embodiments of the ionizable lipid, R 2c is octyl.
  • R 2 is –(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl. In some embodiments of the ionizable lipid, R 2 is –(C1-6 alkylene)-(cyclohexyl)-C1-6 alkyl. In some embodiments of the ionizable lipid, R 2 is –(C 1-6 alkylene)-(cyclopentyl)-C 1-6 alkyl.
  • Variables R 3 , R 3a , R 3b , and R 3c In some embodiments of the ionizable lipid, R 3 is . In some embodiments of the ionizable lipid, R 3a is hydrogen.
  • R 3a is methyl. In some embodiments of the ionizable lipid, R 3a is ethyl. In some embodiments of the ionizable lipid, R 3a is propyl. In some embodiments of the ionizable lipid, R 3a is butyl. In some embodiments of the ionizable lipid, R 3a is pentyl. In some embodiments of the ionizable lipid, R 3a is hexyl. In some embodiments of the ionizable lipid, R 3a is heptyl. In some embodiments of the ionizable lipid, R 3a is octyl.
  • R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3b is methyl. In some embodiments of the ionizable lipid, R 3b is ethyl. In some embodiments of the ionizable lipid, R 3b is propyl. In some embodiments of the ionizable lipid, R 3b is butyl. In some embodiments of the ionizable lipid, R 3b is pentyl. In some embodiments of the ionizable lipid, R 3b is hexyl. In some embodiments of the ionizable lipid, R 3b is heptyl.
  • R 3b is octyl. In some embodiments of the ionizable lipid, R 3a is octyl and R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3a is ethyl and R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3a is hexyl and R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3c is methyl. In some embodiments of the ionizable lipid, R 3c is ethyl. In some embodiments of the ionizable lipid, R 3c is propyl.
  • R 3c is butyl. In some embodiments of the ionizable lipid, R 3c is pentyl. In some embodiments of the ionizable lipid, R 3c is hexyl. In some embodiments of the ionizable lipid, R 3c is heptyl. In some embodiments of the ionizable lipid, R 3c is octyl.
  • variables o, R 1 , R N , R N’ , R N’ , m, n, M, M’, R 2 , R 2a , R 2b , R 2c , R 3 , R 3a , R 3b , and R 3c can each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o,.R 1 , R N , R N’ , R N’ , m, n, M, M’, R 2 , R 2a , R 2b , R 2c , R 3 , R 3a , R 3b , and R 3c can be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R 1 , R N , R N’ , R N’ , m, n, M, M’, R 2 , R 2a , R 2b , R 2c , R 3
  • the ionizable lipid is a compound selected from: In some embodiments, the ionizable lipid is In some embodiments, the ionizable lipid is In some embodiments, the ionizable lipid is Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
  • ionizable lipids are susceptible to the formation of lipid-polynucleotide adducts.
  • ionizable lipids that comprise a tertiary amine group may decompose into one or both of a secondary amine and a reactive aldehyde species capable of interacting with polynucleotides (such as mRNA) to form an ionizable lipid-polynucleotide adduct impurity that can be detected by reverse phase ion pair chromatography (RP-IP HPLC).
  • RP-IP HPLC reverse phase ion pair chromatography
  • the ionizable lipid-polynucleotide adduct impurity is an aldehyde-mRNA adduct impurity. It also has been determined that such adducts may disrupt mRNA translation and impact the activity of lipid nanoparticle (LNP) formulated mRNA products.
  • LNP lipid nanoparticle
  • LNP compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity such as wherein less than about 20%, less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid- polynucleotide adduct impurity, as may be measured by RP-IP HPLC.
  • an LNP composition wherein less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid-polynucleotide adduct impurity, including less than 10%, less than 5%, or less than 1%, as may be measured by RP-IP HPLC.
  • an amount of lipid aldehydes in the composition is less than about 50 ppm, including less than 50 ppm.
  • an amount of N- oxide compounds in the composition is less than about 50 ppm, including less than 50 ppm.
  • an amount of transition metals, such as Fe, in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of alkyl halide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of anhydride compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of ketone compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of conjugated diene compounds in the composition is less than about 50 ppm, including less than 50 ppm.
  • the composition is stable against the formation of ionizable lipid- polynucleotide adduct impurity.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when stored at a temperature of about 25 °C or below, including at an average rate of less than 2% per day.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a temperature of about 5 °C or below, including at an average rate of less than 0.5% per day.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a refrigerated temperature, optionally wherein the refrigerated temperature is about 5 °C.
  • Lipid vehicle (e.g., LNP) compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity can be prepared by methods that inhibit formation of one or both of N-oxides and aldehydes.
  • Such methods may comprise treating a composition comprising an ionizable lipid comprising a tertiary amine group to inhibit formation of one or both of N-oxides and aldehydes, such as by treating the composition with a reducing agent; treating the composition with a chelating agent; adjusting the pH of the composition; adjusting the temperature of the composition; and adjusting the buffer in the composition.
  • Such methods may comprise, prior to combining the ionizable lipid with a polynucleotide, one or more of treating the ionizable lipid with a scavenging agent; treating the ionizable lipid with a reductive treatment agent; treating the ionizable lipid with a reducing agent; treating the ionizable lipid with a chelating agent; treating the polynucleotide with a reducing agent; and treating the polynucleotide with a chelating agent.
  • the scavenging agent, reductive treatment agent, and/or reducing agent may be an agent that reacts with aldehyde, ketone, anhydride and/or diene compounds.
  • a scavenging agent may comprise one or more selected from (O-(2,3,4,5,6- Pentafluorobenzyl)hydroxylamine hydrochloride) (PFBHA), methoxyamine (e.g., methoxyamine hydrochloride), benzyloxyamine (e.g., benzyloxyamine hydrochloride), ethoxyamine (e.g., ethoxyamine hydrochloride), 4-[2-(aminooxy)ethyl]morpholine dihydrochloride, butoxyamine (e.g., tert-butoxyamine hydrochloride), 4-Dimethylaminopyridine (DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), Triethyl
  • DMAP 1,4-d
  • a reductive treatment agent may comprise a boron compound (e.g., sodium borohydride and/or bis(pinacolato)diboron).
  • a reductive treatment agent may comprise a boron compound, such as one or both of sodium borohydride and bis(pinacolato)diboron).
  • a chelating agent may comprise immobilized iminodiacetic acid.
  • a reducing agent may comprise an immobilized reducing agent, such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof.
  • an immobilized reducing agent such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof.
  • a reducing agent may comprise a free reducing agent, such as potassium metabisulfite, sodium thioglycolate, tris(2-carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetyl cysteine, glutathione, dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, lipoic acid, or a combination thereof.
  • the pH may be, or adjusted to be, a pH of from about 7 to about 9 (for example, about 7, about 7.5, about 8, about 8.5, or about 9).
  • a buffer may be selected from sodium phosphate, sodium citrate, sodium succinate, histidine, histidine-HCl, sodium malate, sodium carbonate, and TRIS (tris(hydroxymethyl)aminomethane).
  • a buffer may be TRIS and may be, or adjusted to be, from about 20 mM to about 150 mM TRIS.
  • the temperature of the composition may be, or adjusted to be, 25 0C or less.
  • the composition may also comprise a free reducing agent or antioxidant.
  • Non-cationic lipids In certain embodiments, a lipid nanoparticle described herein comprise one or more non- cationic lipids.
  • Non-cationic lipids may be phospholipids.
  • a lipid nanoparticle comprises 5-25 mol% non-cationic lipid.
  • a lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid.
  • a lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.
  • a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phospho
  • a lipid nanoparticle comprises 5 – 15 mol%, 5 – 10 mol%, or 10 – 15 mol% DSPC.
  • a lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.
  • the lipid composition of a lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., an mRNA) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a membrane e.g., a cellular or intracellular membrane.
  • elements e.g., an mRNA
  • a lipid-containing composition e.g., LNPs
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid of the present disclosure comprises 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC),
  • a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC.
  • a phospholipid useful or potentially useful in the present disclosure is a compound of Formula (IX): or a salt thereof, wherein: each R 1 is independently optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R 1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula: each instance of L 2 is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C 1-6 alkylene is optionally replaced with O, N(R N ), S
  • the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT/US2018/037922.
  • a lipid nanoparticle comprises 5-25 mol% non-cationic lipid relative to the other lipid components.
  • a lipid nanoparticle may comprise 5-30 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, or 25-30 mol% non-cationic lipid.
  • a lipid nanoparticle comprises a 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or 30 mol% non- cationic lipid. In some embodiments, a lipid nanoparticle comprises 5-25 mol% phospholipid relative to the other lipid components. For example, the lipid nanoparticle may comprise 5-30 mol%, 5- 15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, or 25-30 mol% phospholipid.
  • the lipid nanoparticle 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or 30 mol% phospholipid lipid.
  • Structural Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid includes sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in a lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • “sterols” are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol.
  • the structural lipids may be one or more of the structural lipids described in U.S. Application No.16/493,814.
  • a lipid nanoparticle comprises 25-55 mol% structural lipid relative to the other lipid components.
  • a lipid nanoparticle may comprise 10- 55 mol%, 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid.
  • a lipid nanoparticle comprises 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid.
  • a lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 34-35 mol%, 35- 36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%.
  • a lipid nanoparticle comprises 25-55 mol% sterol.
  • a lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol.
  • a lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol. In some embodiments, a lipid nanoparticle comprises 35-40 mol% cholesterol. For example, a lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.
  • PEG polyethylene Glycol
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids.
  • PEG-lipid or “PEG-modified lipid” refers to polyethylene glycol (PEG)-modified lipids.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3- amines.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3- amines.
  • PEGylated lipids PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine
  • the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG-modified lipid is PEG- DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and/or PEG-DPG.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16.
  • a PEG moiety for example an mPEG-NH2
  • the PEG-lipid is PEG 2k -DMG.
  • a lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • some of the other lipid components (e.g., PEG lipids) of various formulae described herein may be synthesized as described International Patent Application No. PCT/US2016/000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.
  • the lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-modified lipids are a modified form of PEG DMG.
  • PEG- DMG has the following structure:
  • PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy- PEGylated lipid comprises an –OH group at the terminus of the PEG chain.
  • a PEG lipid useful in the present disclosure is a compound of Formula (X): (X), or salts thereof, wherein: R 3 is –OR O ; R O is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is optionally substituted C 1-10 alkylene, wherein at least one methylene of the optionally substituted C 1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ); D is a moiety obtained by click chemistry or a moiety cleavable under
  • the compound of Fomula (X) is a PEG-OH lipid (i.e., R 3 is – OR O , and R O is hydrogen).
  • the compound of Formula (X) is of Formula (X-OH): (X-OH), or a salt thereof.
  • a PEG lipid useful in the present disclosure is a PEGylated fatty acid.
  • a PEG lipid useful in the present disclosure is a compound of Formula (XI).
  • R 3 is–OR O ;
  • R O is hydrogen, optionally substituted alkyl or an oxygen protecting group;
  • r is an integer between 1 and 100, inclusive;
  • the compound of Formula (XI) is of Formula (XI-OH): or a salt thereof.
  • r is 40-50.
  • the compound of Formula (XI) is: . or a salt thereof.
  • the compound of Formula (XI) is In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.
  • the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. US15/674,872.
  • a lipid nanoparticle comprises 0.5-15 mol% PEG lipid relative to the other lipid components.
  • a lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% PEG lipid.
  • a lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG- lipid.
  • a lipid nanoparticle comprises 1-5% PEG-modified lipid, optionally 1-3 mol%, for example, 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%.
  • a lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid.
  • a lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%.
  • a lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.
  • Some embodiments comprise adding PEG to a composition comprising an LNP encapsulating a nucleic acid (e.g., which already includes PEG in the amounts listed above). Without being bound by theory, it is believed that spiking an LNP composition with additional PEG can provide benefits during lyophilization.
  • a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.
  • an LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.
  • an LNP of the present disclosure comprises an ionizable amino lipid of any of Formula VI, VII or VIIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.
  • an LNP of the present disclosure comprises an ionizable amino lipid of any of Formula VI, VII or VIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula XI.
  • an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula VIII, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI.
  • an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula IX, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI.
  • an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid having Formula IX, a structural lipid, and a PEG lipid comprising a compound having Formula XI.
  • a lipid nanoparticle comprises 49 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
  • a lipid nanoparticle comprises 49 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.
  • a lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
  • an LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1.
  • an LNP of the present disclosure comprises an N:P ratio of about 6:1.
  • an LNP of the present disclosure comprises an N:P ratio of about 3:1, 4:1, or 5:1.
  • an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1.
  • an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1. In some embodiments, an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1. Some embodiments comprise a composition having one or more LNPs having a diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less.
  • Some embodiments comprise a composition having a mean LNP diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less.
  • the composition has a mean LNP diameter from about 30nm to about 150nm, or a mean diameter from about 60nm to about 120nm.
  • An LNP may comprise or one or more types of lipids, including but not limited to amino lipids (e.g., ionizable amino lipids), neutral lipids, non-cationic lipids, charged lipids, PEG- modified lipids, phospholipids, structural lipids and sterols.
  • the composition comprises a liposome.
  • a liposome is a lipid particle comprising lipids arranged into one or more concentric lipid bilayers around a central region. The central region of a liposome may comprise an aqueous solution, suspension, or other aqueous composition.
  • a lipid nanoparticle may comprise two or more components (e.g., amino lipid and nucleic acid, PEG-lipid, phospholipid, structural lipid).
  • a lipid nanoparticle may comprise an amino lipid and a nucleic acid.
  • Compositions comprising lipid nanoparticles, such as those described herein, may be used for a wide variety of applications, including the stealth delivery of mRNA with minimal adverse innate immune response. Effective in vivo delivery of nucleic acids represents a continuing medical challenge. Exogenous nucleic acids (i.e., originating from outside of a cell or organism) are readily degraded in the body, e.g., by the immune system.
  • a particulate carrier e.g., lipid nanoparticles
  • the particulate carrier should be formulated to have minimal particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids intracellularly, and illicit no or minimal immune response.
  • many conventional particulate carriers have relied on the presence and/or concentration of certain components (e.g., PEG-lipid).
  • certain components e.g., PEG-lipid
  • certain components may decrease the stability of encapsulated nucleic acids (e.g., mRNA molecules). The reduced stability may limit the broad applicability of the particulate carriers.
  • lipid nanoparticles comprise one or more of ionizable molecules, polynucleotides, and optional components, such as structural lipids, sterols, neutral lipids, phospholipids and a molecule capable of reducing particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipid), such as those described above.
  • an LNP described herein may include one or more ionizable molecules (e.g., amino lipids or ionizable lipids).
  • the ionizable molecule may comprise a charged group and may have a certain pKa.
  • the pKa of the ionizable molecule may be greater than or equal to about 6, greater than or equal to about 6.2, greater than or equal to about 6.5, greater than or equal to about 6.8, greater than or equal to about 7, greater than or equal to about 7.2, greater than or equal to about 7.5, greater than or equal to about 7.8, greater than or equal to about 8.
  • the pKa of the ionizable molecule may be less than or equal to about 10, less than or equal to about 9.8, less than or equal to about 9.5, less than or equal to about 9.2, less than or equal to about 9.0, less than or equal to about 8.8, or less than or equal to about 8.5. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 6 and less than or equal to about 8.5). Other ranges are also possible. In embodiments in which more than one type of ionizable molecule are present in a particle, each type of ionizable molecule may independently have a pKa in one or more of the ranges described above.
  • an ionizable molecule comprises one or more charged groups.
  • an ionizable molecule may be positively charged or negatively charged.
  • an ionizable molecule may be positively charged.
  • an ionizable molecule may comprise an amine group.
  • the term “ionizable molecule” has its ordinary meaning in the art and may refer to a molecule or matrix comprising one or more charged moiety.
  • a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc.
  • the charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).
  • positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups.
  • the charged moieties comprise amine groups.
  • negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged.
  • the charge density of the molecule and/or matrix may be selected as desired.
  • an ionizable molecule e.g., an amino lipid or ionizable lipid
  • the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above.
  • the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively.
  • an amide which can be hydrolyzed to form an amine, respectively.
  • Those of ordinary skill in the art will be able to determine whether a given chemical moiety carries a formal electronic charge (for example, by inspection, pH titration, ionic conductivity measurements, etc.), and/or whether a given chemical moiety can be reacted (e.g., hydrolyzed) to form a chemical moiety that carries a formal electronic charge.
  • the ionizable molecule e.g., amino lipid or ionizable lipid
  • the molecular weight of an ionizable molecule is less than or equal to about 2,500 g/mol, less than or equal to about 2,000 g/mol, less than or equal to about 1,500 g/mol, less than or equal to about 1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equal to about 900 g/mol, less than or equal to about 800 g/mol, less than or equal to about 700 g/mol, less than or equal to about 600 g/mol, less than or equal to about 500 g/mol, less than or equal to about 400 g/mol, less than or equal to about 300 g/mol, less than or equal to about 200 g/mol, or less than or equal to about 100 g/mol.
  • the molecular weight of an ionizable molecule is greater than or equal to about 100 g/mol, greater than or equal to about 200 g/mol, greater than or equal to about 300 g/mol, greater than or equal to about 400 g/mol, greater than or equal to about 500 g/mol, greater than or equal to about 600 g/mol, greater than or equal to about 700 g/mol, greater than or equal to about 1000 g/mol, greater than or equal to about 1,250 g/mol, greater than or equal to about 1,500 g/mol, greater than or equal to about 1,750 g/mol, greater than or equal to about 2,000 g/mol, or greater than or equal to about 2,250 g/mol.
  • each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above.
  • the percentage (e.g., by weight, or by mole) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and/or of all the ionizable molecules within a particle may be greater than or equal to about 15%, greater than or equal to about 16%, greater than or equal to about 17%, greater than or equal to about 18%, greater than or equal to about 19%, greater than or equal to about 20%, greater than or equal to about 21%, greater than or equal to about 22%, greater than or equal to about 23%, greater than or equal to about 24%, greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 35%, greater than or equal to about 40%, greater than or equal to about 42%, greater than or equal to about 45%, greater than or equal to about 48%, greater than or equal to about 50%, greater than or equal to about 52%, greater than or equal to about 55%, greater than or equal to about 58%, greater than
  • the percentage (e.g., by weight, or by mole) may be less than or equal to about 70%, less than or equal to about 68%, less than or equal to about 65%, less than or equal to about 62%, less than or equal to about 60%, less than or equal to about 58%, less than or equal to about 55%, less than or equal to about 52%, less than or equal to about 50%, or less than or equal to about 48%. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 20% and less than or equal to about 60%, greater than or equal to 40% and less than or equal to about 55%, etc.).
  • each type of ionizable molecule may independently have a percentage (e.g., by weight, or by mole) in one or more of the ranges described above.
  • the percentage e.g., by weight, or by mole
  • the percentage may be determined by extracting the ionizable molecule(s) from the dried particles using, e.g., organic solvents, and measuring the quantity of the agent using high pressure liquid chromatography (i.e., HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS).
  • HPLC may be used to quantify the amount of a component, by, e.g., comparing the area under the curve of a HPLC chromatogram to a standard curve.
  • charge or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule.
  • partial negative charge and “partial positive charge” are given their ordinary meaning in the art.
  • a “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom.
  • compositions for use to induce an immune response in a subject e.g., as vaccines against a flavivirus or multiple flaviviruses.
  • the flavivirus is selected from the group consisting of: Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus and Omsk virus.
  • the flavivirus is Dengue virus.
  • the flavivirus is Zika virus.
  • the flavivirus is Dengue virus and Zika virus. Multivalent Vaccines
  • the compositions may include single mRNA molecules or multiple RNAs (e.g., mRNAs) encoding two or more antigens of the same or different species.
  • composition includes an mRNA or multiple RNAs (e.g., mRNAs) encoding two or more flavivirus proteins.
  • the mRNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more flavivirus (e.g., DENV) proteins.
  • the composition comprises a first mRNA encoding a first stabilized heterotetramer and a second mRNA encoding a second stabilized heterotetramer.
  • the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the first and second stabilized heterotetramers are of the same DENV serotype.
  • the first and second stabilized heterotetramers are of different DENV serotypes.
  • the composition comprises a first mRNA encoding a first stabilized heterotetramer, a second mRNA encoding a second stabilized heterotetramer, and a third mRNA encoding a third stabilized heterotetramer.
  • the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the third stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the first, second, and third stabilized heterotetramers are of the same DENV serotype. In some embodiments, the first and second stabilized heterotetramers are of the same DENV serotype and the third stabilized heterotetramer is of a different DENV serotype. In some embodiments, the first, second, and third stabilized heterotetramers are of different DENV serotypes.
  • the composition comprises a first mRNA encoding a stabilized DENV1 heterotetramer, a second mRNA encoding a stabilized DENV2 heterotetramer, a third mRNA encoding a stabilized DENV3 heterotetramer, and a fourth mRNA encoding a stabilized DENV4 heterotetramer.
  • two or more different mRNA encoding antigens may be formulated in the same lipid nanoparticle.
  • two or more different mRNA encoding antigens may be formulated in separate lipid nanoparticles (each mRNA formulated in a single lipid nanoparticle).
  • Lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNAs (e.g., mRNAs) encoding multiple antigens) or may be administered separately.
  • a single vaccine composition e.g., comprising multiple RNAs (e.g., mRNAs) encoding multiple antigens
  • Some aspects relate to multivalent vaccines that comprise components to protect a subject against more than one flavivirus.
  • Multivalent flavivirus vaccines can include two (bivalent), three (trivalent), four (quadrivalent), five (pentavalent), or more components that each independently are designed to protect against one of a variety of flaviviruses.
  • a multivalent vaccine comprises two mRNAs, each directed to a different serotype of a flavivirus (e.g., Dengue virus).
  • the mRNAs are present at a 1:1, 2:1, 4:1, 1:1:1, 2:1:1, 4:2:1, 1:1:1:1, 2:1:1:1, 4:2:1:1, or 4:2:2:1 mass ratio or a 1:1, 2:1, 4:1, 1:1:1, 2:1:1, 4:2:1, 1:1:1:1, 2:1:1:1, 4:2:1:1, or 4:2:2:1 molar ratio.
  • Combination Vaccines Some embodiments of vaccines include combination vaccines.
  • a "combination vaccine" refers to a vaccine comprising two or more components for eliciting an immune response against multiple flaviviruses.
  • the combination vaccine elicits an immune response against Dengue virus and Zika virus (e.g., an immune response agains DENV1, DENV2, DENV3, DENV4, and/or ZIKV).
  • a combination vaccine includes one or more RNAs encoding an antigen of more than one flavivirus.
  • a combination vaccine may comprise mRNAs encoding stabilized E and M protein heterotetramers from DENV1, DENV2, DENV3, DENV4, and ZIKV.
  • the composition comprises a first mRNA encoding a first stabilized heterotetramer and a second mRNA encoding a second stabilized heterotetramer.
  • the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the second stabilized heterotetramer is a ZIKV stabilized heterotetramer.
  • the composition comprises a first mRNA encoding a first stabilized heterotetramer, a second mRNA encoding a second stabilized heterotetramer, and a third mRNA encoding a third stabilized heterotetramer.
  • the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the third stabilized heterotetramer is a ZIKV stabilized heterotetramer.
  • the first and second stabilized heterotetramers are of the same DENV serotype. In some embodiments, the first and second stabilized heterotetramers are of different DENV serotypes.
  • the composition comprises a first mRNA encoding a first stabilized heterotetramer, a second mRNA encoding a second stabilized heterotetramer, a third mRNA encoding a third stabilized heterotetramer, and fourth mRNA encoding a fourth stabilized heterotetramer.
  • the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the third stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer.
  • the fourth stabilized heterotetramer is a ZIKV stabilized heterotetramer.
  • the first, second, and third stabilized heterotetramers are of the same DENV serotype.
  • the first and second stabilized heterotetramers are of the same DENV serotype and the third stabilized heterotetramer is of a different DENV serotype.
  • the first, second, and third stabilized heterotetramers are of different DENV serotypes.
  • the composition comprises a first mRNA encoding a stabilized DENV1 heterotetramer, a second mRNA encoding a stabilized DENV2 heterotetramer, a third mRNA encoding a stabilized DENV3 heterotetramer, a fourth mRNA encoding a stabilized DENV4 heterotetramer, and a fifth mRNA encoding a stabilized ZIKV heterotetramer.
  • the mRNAs are present in various ratios in order to induce efficacious immune responses.
  • the ratio of mRNA encoding DENV to mRNA encoding ZIKV is a 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 molar or mass ratio.
  • the mRNA encoding the DENV serotype heterotetramers e.g., DENV1, DENV2, DENV3, and DENV4 are present at a 1:1, 2:1, 4:1, 1:1:1, 2:1:1, 4:2:1, 1:1:1:1, 2:1:1:1, 4:2:1:1, or 4:2:2:1 molar or mass ratio.
  • the nucleotides encoding the stabilized heterotetramers are encapsulated in lipid nanoparticles for delivery.
  • one or more RNAs encoding polypeptides from different flaviviruses are encapsulated in a single lipid nanoparticle.
  • each RNA encoding a polypeptide from a single flavivirus is encapsulated in separate lipid nanoparticles. These nanoparticles can then be combined into a single vaccine composition or administered separately.
  • compositions e.g., pharmaceutical compositions, such as vaccines
  • methods, kits and reagents for prevention of flavivirus infections and other conditions directly or indirectly cause by flavivirus infection in humans and other mammals for example.
  • the compositions are protein vaccines, DNA vaccines or mRNA vaccines.
  • the compositions containing mRNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the mRNA are translated in vivo to produce an antigenic polypeptide (antigen).
  • an “effective amount” of a composition is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the mRNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject.
  • an effective amount of a composition provides an immune response as a function of antigen administration to a subject or production in the cells of the subject.
  • an effective amount of the composition containing mRNA having at least one chemical modification are more efficient than a composition containing a corresponding 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 mRNA vaccine), increased protein translation and/or expression 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.
  • composition refers to the combination of an active agent (e.g., mRNA) with a carrier (e.g., lipid composition, e.g., LNP)), inert or active, making the composition especially suitable for prophylactic use in vivo or ex vivo.
  • a carrier e.g., lipid composition, e.g., LNP
  • a “pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects.
  • the carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it.
  • One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent.
  • compositions comprising polynucleotides and their encoded polypeptides in accordance with the present disclosure may be used for the prevention of a flavivirus infection.
  • a composition may be administered prophylactically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase.
  • the amount of mRNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.
  • a composition may be administered with other prophylactic or therapeutic compounds.
  • a prophylactic or therapeutic compound may be an adjuvant or a booster.
  • 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, 12 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, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more.
  • 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, or 6 months.
  • the booster may comprise the same or different mRNA as compared to the earlier administration of the prophylactic composition.
  • the booster in some embodiments is monovalent (e.g., the mRNA encodes a single antigen). In some embodiments, the booster is multivalent (e.g., the mRNA encodes more than one antigen).
  • “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful.
  • a composition disclosed herein is administered to a subject enterally.
  • an enteral administration of the composition is oral.
  • a composition disclosed herein is administered to the subject parenterally.
  • a composition disclosed herein is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs.
  • the administration is intramuscular.
  • a composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need.
  • pharmaceutical compositions including mRNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.
  • the mRNA may be formulated or administered alone or in conjunction with one or more other components.
  • a vaccine may comprise other components including, but not limited to, adjuvants.
  • a vaccine does not include an adjuvant (it is adjuvant free).
  • An mRNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients.
  • vaccines comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both.
  • Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free.
  • a vaccine is administered to humans, human patients or subjects.
  • active ingredient generally refers to the mRNA contained therein, for example, mRNA encoding a stabilized heterotetramer.
  • Formulations of the vaccines described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • such preparatory methods include the step of bringing the active ingredient (e.g., 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.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure 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.
  • the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • compositions e.g., vaccines
  • methods, kits and reagents for inducing an immune response in a subject e.g., an immune response to the composition
  • a subject may be any mammal, including non-human primate and human subjects.
  • a subject is a human subject.
  • a composition is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen-specific immune response.
  • the mRNA encoding the flavivirus protein is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.
  • Prophylactic protection from other conditions cause directly or indirectly by flavivirus infection can be achieved following administration of a composition of the present disclosure.
  • the compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the composition once (optionally followed by a single booster). Dosing may need to be adjusted accordingly.
  • a method of eliciting an immune response in a subject against a flavivirus protein (or multiple antigens) is provided in aspects of the present disclosure.
  • a method involves administering to the subject a vaccine comprising a mRNA having an open reading frame encoding a flavivirus protein (or multiple antigens), thereby inducing in the subject an immune response specific to the flavivirus protein (or multiple antigens), wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti- antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen.
  • An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen.
  • a prophylactically effective dose is an effective dose that prevents infection with the virus and/or reduces symptoms or negative outcomes from a viral infection at a clinically acceptable level.
  • the effective dose is a dose listed in a package insert for the vaccine
  • the subject heterotetrameric vaccines are non-infectious and can be administered as proteins or nucleic acids (e.g., mRNAs).
  • a composition of the present disclosure may be administered by any route that results in a prophylactically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration.
  • the present disclosure provides methods comprising administering the subject 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.
  • the effective amount of the mRNA may be as low as 20 ⁇ g, administered for example as a single dose or as two 10 ⁇ g doses (e.g., a first effective vaccine dose and a second effective vaccine dose).
  • the first effective vaccine dose and the second effective vaccine dose are the same amount.
  • the first effective vaccine dose and the second effective vaccine dose are different amounts.
  • the effective amount is a total dose of 5 ⁇ g- 30 ⁇ g, 5 ⁇ g -25 ⁇ g, 5 ⁇ g -20 ⁇ g, 5 ⁇ g -15 ⁇ g, 5 ⁇ g -10 ⁇ g, 10 ⁇ g -30 ⁇ g, 10 ⁇ g -25 ⁇ g, 10 ⁇ g- 20 ⁇ g, 10 ⁇ g -15 ⁇ g, 15 ⁇ g -30 ⁇ g, 15 ⁇ g -25 ⁇ g, 15 ⁇ g -20 ⁇ g, 20 ⁇ g -30 ⁇ g, 25 ⁇ g -30 ⁇ g, or 25 ⁇ g-300 ⁇ g.
  • the effective dose (e.g., effective amount) is at least 10 ⁇ g and less than 25 ⁇ g of the composition. In some embodiments, the effective dose (e.g., effective amount) is at least 5 ⁇ g and less than 25 ⁇ g of the composition.
  • the effective amount may be a total dose of 5 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g, 200 ⁇ g, 250 ⁇ g, or 300 ⁇ g.
  • the effective amount (e.g., effective dose) is a total dose of 10 ⁇ g. In some embodiments, the effective amount is a total dose of 20 ⁇ g (e.g., two 10 ⁇ g doses). In some embodiments, the effective amount is a total dose of 25 ⁇ g. In some embodiments, the effective amount is a total dose of 30 ⁇ g. In some embodiments, the effective amount is a total dose of 50 ⁇ g. In some embodiments, the effective amount is a total dose of 60 ⁇ g (e.g., two 30 ⁇ g doses). In some embodiments, the effective amount is a total dose of 75 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g.
  • the effective amount is a total dose of 150 ⁇ g. In some embodiments, the effective amount is a total dose of 200 ⁇ g. In some embodiments, the effective amount is a total dose of 250 ⁇ g. In some embodiments, the effective amount is a total dose of 300 ⁇ g.
  • the mRNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). In some embodiments the composition or vaccine is administered to a subject intramuscularly.
  • compositions e.g., RNA vaccines
  • the mRNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a flavivirus antigen).
  • an effective amount is a dose of the mRNA effective to produce an antigen-specific immune response.
  • methods of inducing an antigen-specific immune response in a subject are characterized by measuring an anti-DENV antigen antibody titer (e.g., neutralization titer) produced in a subject administered a composition as provided herein.
  • an anti-DENV antigen antibody titer e.g., neutralization titer
  • the antigen-specific immune response is characterized by measuring an anti-ZIKV antigen antibody titer (e.g., neutralization titer) produced in a subject administered a composition as provided herein.
  • the antigen-specific immune response is characterized by measuring an anti-YFV antigen antibody titer (e.g., neutralization titer) produced in a subject administered a composition 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 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 is a common assay for determining antibody titers, for example.
  • Other Embodiments include: Embodiment 1. A messenger ribonucleic acid (mRNA) vaccine, comprising: an mRNA comprising an open reading frame encoding a modified dengue virus (DENV) protein, relative to a corresponding naturally occurring DENV protein; and a lipid nanoparticle.
  • mRNA messenger ribonucleic acid
  • DENV modified dengue virus
  • Embodiment 3 Embodiment 3.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA comprising an open reading frame encoding a Zika virus (ZIKV) protein, relative to a corresponding naturally occurring ZIKV protein; and a lipid nanoparticle.
  • mRNA vaccine comprising: an mRNA comprising an open reading frame encoding a Zika virus (ZIKV) protein, relative to a corresponding naturally occurring ZIKV protein; and a lipid nanoparticle.
  • the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352.
  • Embodiment 5 Embodiment 5.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA comprising an open reading frame encoding a Yellow fever virus (YFV) protein, relative to a corresponding naturally occurring YFV protein; and a lipid nanoparticle.
  • mRNA messenger ribonucleic acid
  • Embodiment 6 The mRNA vaccine of embodiment 5, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA comprising an open reading frame encoding a dengue virus (DENV) protein, relative to a corresponding naturally occurring DENV protein; an mRNA comprising an open reading frame encoding a Zika virus (ZIKV) protein, relative to a corresponding naturally occurring ZIKV protein; and a lipid nanoparticle.
  • DENV dengue virus
  • ZIKV Zika virus
  • Embodiment 10 The mRNA vaccine of any one of embodiments 6-9, further comprising an mRNA comprising an open reading frame encoding a Yellow fever virus (YFV) protein, relative to a corresponding naturally occurring YFV protein; and a lipid nanoparticle.
  • YFV Yellow fever virus
  • Embodiment 12. The mRNA vaccine of any one of the preceding embodiments, wherein the one or more mRNA(s) of the vaccine comprises a chemical modification.
  • Embodiment 13 The mRNA vaccine of embodiment 12, wherein 100% of the uracil nucleotides of the one or more mRNA(s) comprise a chemical modification.
  • Embodiment 15 The mRNA vaccine of any one of the preceding embodiments, wherein the lipid nanoparticle comprises an ionizable lipid, a neutral lipid, a sterol, and a PEG-modified lipid.
  • Embodiment 16. The mRNA vaccine of embodiment 15, wherein the lipid nanoparticle comprises 40–50 mol% ionizable lipid, 5–15 mol% neutral lipid, 30–50 mol% sterol, and 0.5–3 mol% PEG-modified lipid.
  • Embodiment 18 A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject one or more doses of the mRNA vaccine of any one of the preceding embodiments in an effective amount to produce an immune response to the protein.
  • Embodiment 19 The method of embodiment 18, wherein the vaccine is administered intramuscularly.
  • Embodiment 20 The method of embodiment 18 or 19 comprising administering a single dose of the vaccine to the subject.
  • Embodiment 21 The method of embodiment 18 or 19 comprising administering a prime dose and a booster dose of the vaccine to the subject.
  • Embodiment 22 A protein comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363.
  • Embodiment 23 A protein comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170- 172, 177-178, 212-213, 285-301, and 347-352.
  • a protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214.
  • Embodiment 25 A fusion protein comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein: (a) the E protein comprises at least one substitution at one or more positions selected from: (i) T76, R93, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to SEQ ID NOs: 274, 276, or 280 ; or (ii) T76, K93, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A
  • Embodiment 26 The fusion protein of embodiment 25, wherein the fusion protein comprises, from N-terminus to C-terminus, the E protein and the M protein.
  • Embodiment 27 The fusion protein of embodiment 25, wherein the fusion protein comprises, from N-terminus to C-terminus, the M protein and the E protein.
  • Embodiment 28 The fusion protein of any one of embodiments 25-27, further comprising a precursor (Pr) protein.
  • Embodiment 29 The fusion protein of embodiment 28, wherein the fusion protein comprises, from N-terminus to C-terminus, the Pr protein, the M protein and the E protein.
  • Embodiment 30 The fusion protein of any one of embodiments 25-29, further comprising a linker.
  • Embodiment 31 The fusion protein of any one of embodiments 25-29, further comprising a linker.
  • Embodiment 32. The fusion protein of any one of embodiments 30-31, wherein the E protein and the M protein are linked by the linker.
  • a messenger ribonucleic acid (mRNA) vaccine capable of inducing an immune response against a flavivirus antigen
  • the mRNA vaccine comprises an mRNA polynucleotide having an open reading frame (ORF) encoding a fusion protein
  • the fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 273-281
  • the fusion protein comprises a flavivirus envelope (E) protein and a flavivirus membrane (M) protein
  • the E protein comprises at least one substitution at one or more positions selected from: (i) T76, R93, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to SEQ ID NOs
  • Embodiment 34 The mRNA vaccine of embodiment 33, wherein the fusion protein comprises, from N-terminus to C-terminus, the E protein and the M protein.
  • Embodiment 35 The mRNA vaccine of embodiment 33, wherein the fusion protein comprises, from N-terminus to C-terminus, the M protein and the E protein.
  • Embodiment 36 The mRNA vaccine of any one of embodiments 33-35, wherein the fusion protein further comprises a precursor (Pr) protein.
  • Embodiment 37 The mRNA vaccine of embodiment 36, wherein the fusion protein comprises, from N-terminus to C-terminus, the Pr protein, the M protein and the E protein.
  • Embodiment 38 The mRNA vaccine of embodiment 36, wherein the fusion protein comprises, from N-terminus to C-terminus, the Pr protein, the M protein and the E protein.
  • the mRNA vaccine of any one of embodiments 33-40, wherein the fusion protein further comprises a signal peptide.
  • the mRNA vaccine of embodiment 41, wherein the signal peptide comprises an amino acid sequence selected from SEQ ID NOs: 271 or 272.
  • the mRNA vaccine of embodiment 43, wherein the flavivirus is Dengue virus.
  • the mRNA vaccine of embodiment 43, wherein the flavivirus is Zika virus.
  • a method of inducing neutralizing antibodies in a subject by administering a stabilized flavivirus heterotetramer comprising: (a) means for inducing an immune response to the flavivirus in the subject; and (b) a flavivirus envelope (E) protein, wherein the E protein comprises an amino acid substitution at each of position 76 and at position 107, wherein the positions are numbered by alignment of the E protein to SEQ ID NO: 276; and a flavivirus membrane (M) protein, wherein administration of the flavivirus heterotetramer has increased neutralization antibody titers compared to wild-type flavivirus prME protein.
  • E flavivirus envelope
  • M flavivirus membrane
  • a stabilized flavivirus heterotetramer comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein
  • the E protein comprises a fusion loop, a bc loop, and a transmembrane region
  • the fusion loop comprises an amino acid substitution at position 107 and the bc loop comprises an amino acid substitution at position 76 and wherein the positions correspond to SEQ ID NO: 276
  • the M protein comprises a N-terminus and a C-terminus; wherein the N-terminus comprises an N- terminal region comprising at least one amino acid substitution relative to the corresponding wild-type flavivirus M protein.
  • VLPs Selected Dengue, Zika, and Yellow Fever Antigens Virus-like particles
  • VLPs Selected Dengue, Zika, and Yellow Fever Antigens Virus-like particles
  • plasmid DNA Prior work has demonstrated that using plasmid DNA is an appropriate surrogate for proteins expressed as mRNA.
  • Each of the immunogens tested comprised either: (i) a Pr protein sequence followed by a membrane (M) protein and followed by an envelope (E) protein (“PrM/E”); (ii) a protein comprising an E protein followed by an M protein (“E/M”); or (iii) a protein comprising an M protein followed by an E protein (“M/E”).
  • VLPs were designed and engineered based on visual inspection of the proteins using graphical software (e.g., chimeraX), and mutations were designed accordingly.
  • pcDNA3.4 plasmids containing the gene sequence for the engineered VLPs were procured from GenScript.
  • Wild-type DENV2 PrM/E and DENV2 M/E VLPs were used as controls (“WT” and “wt”, respectively). Analysis of dot-blot results showed design numbers DENV2-E/M_55, DENV2-E/M_56, DENV2-E/M_57, DENV2-M/E_111, DENV2-M/E_131, DENV2-M/E_133, DENV2-M/E_134, DENV2-M/E_135, and DENV2-M/E_136 displayed significantly higher expression than the WT VLPs when probed with the conformational bnAb which detects the desired E dimer epitope of DENV2.
  • the table below shows that DENV2-M/E_136 gave highest detection with the A11 antibody compared to detection of WT with the same antibody.
  • no signal was observed for DENV2-prME_AS4 with the 4G2 antibody compared to the background, but a signal was observed with the A11 antibody, indicating that this design preferred binding to A11 and has no detectable binding to 4G2.
  • dot-blot expression profiles were performed as described above. Expression was assessed using an A11 bnAb, a 4G2 mAb, or an EDE1 C8 bnAb (Table 6).
  • EDE1 C8 bnAb (“C8”) was used to detect ZIKV VLP designs. Dot-blot analysis using the C8 bnAb demonstrated bright signals in all ZIKV VLP designs except 2-4, indicating recognition by a desired conformational antibody. Designs showing high reactivity to the broadly neutralizing antibodies and low reactivity to the anti-fusion loop antibodies were selected for further analysis. Table 6. ZIKV, DENV1, DENV3 and DENV4 – Relative Expression Data Note: EDE1 C8, specific for DENVs1-4 and ZIKV; EDE2 A11, specific for DENVs1-4; FLE 4G2, anti-fusion loop antibody. Detection levels are comparable only within each virus, and are indicated as relative to their WT version.
  • ZIKV, DENV1, DENV3 and DENV4 Relative Expression Data Note: C10, specific for DENVs1-4 and ZIKV; C8, specific for DENVs1-4 and ZIKV; B7, specific for DENVs1-4; A11, specific for DENVs1-4; SIgN_3C, specific for ZIKV, DENV2, DENV3; MZ4, specific for ZIKV, DENV2, DENV3; ZVDV1IDIII_Z021, specific for ZIKV and DENV1. Table 7B.
  • ZIKV, DENV1, DENV3 and DENV4 – Relative Expression Data (neutralizing antibodies) Note: DV1EDI_1F4, DENV1-specific; ZIKV_195, ZIKV-specific; ZVED_8DV6, ZIKV-specific; 2D22, DENV2-specific; DV2EIII_2C8, DENV2-specific; DV3dim_5J7, DENV3-specific; DV4EDI_5H2, DENV4-specific Table 7C.
  • VLPs of sizes 40 to 60 nm were observed for each construct.
  • Fab C8 binding was observed for DENV2 102 VLPs as protrusions on the smooth surface, while ZIKV 50 VLPs showed binding to Fab C8 in native conditions as observed with cryo-EM.
  • dot-blot expression profiles were performed as described above. Expression was assessed using either specific mAbs 5A (“5A”), or a YD73 mAb, which targets the domain III region of the YFV-E protein (EDIII) (Table 8).
  • YFV – Relative Expression Data Note: 5A, monoclonal antibody against YFV; YD73, anti-EDIII antibody (targets domain III region); tested in duplicate (“1” and “2” in the table below); Brazilian and Asibi strains tested (“Br” and “Asb”, respectively) Further ZIKV and DENV antigens were screened for expression using conformational and linear epitope antibodies (Table 9). In the table below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale: indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Expression was measured by dot-blot analysis as described above.
  • cross-reactive antibodies used i.e., EDE1 C10, EDE1 C8, EDE2 A11, SIgN-3C, and 4G2
  • EDE1 C10, EDE1 C8, EDE2 A11, SIgN-3C, and 4G2 have different binding affinities to all five viruses, thus their signal is used to discriminate expression of designs for the same virus.
  • D1-005, D2-029, D3-002, D4-003, and Z50HANg demonstrated the highest expression and detection among the designs for the five different viruses. Table 9.
  • EDE1 C10 binds to ZIKV and DENVs1-4
  • EDE1 C8 binds to ZIKV and DENVs1-4
  • EDE2 A11 binds to DENVs1-4
  • DV4 EDI 5H2 binds to DENV4
  • SIgN-3C binds to ZIKV and DENVs1-4
  • DV3_5J7 binds to DENV3
  • 2D22 binds to DENV2
  • 4G2 anti-fusion loop antibody VLPs showing the highest expression levels as measured by dot-blot analysis were chosen for purification and further protein analysis.
  • FIG.3 demonstrates the results of one of the candidates, ZIKV_HA_50_E/M VLPs, which were expressed in Expi293 cells then purified as described in “Methods” below. Further purification was then performed using streptavidin- tagged EDE1 Fab C8 that bound to the VLPs. The antibody-VLP complexes were captured by a streptavidin column, and VLPs were then eluted. These purified VLPs were then tested using SDS-PAGE in either non-reducing (-BME) or reducing (+BME) conditions (FIG.3, columns 2 and 3, respectively).
  • the ZIKV_HA_50_E/M VLPs contain an engineered disulfide bond that stabilizes an (EM)2 heterotetramer, which is reduced in the presence of BME.
  • FIG.3 demonstrates the presence of intact ZIKV_HA_50_E/M heterotetramers at ⁇ 130 kDa in non- reducing conditions (column 2) and heterodimers at ⁇ 65 kDa in reducing conditions (column 3), verifying the presence of a ZIKV_HA_50_E/M heterotetrameric complex containing the engineered disulfide bond.
  • Additional ZIKV, DENV2, and DENV3 antigens were screened for expression using conformational and linear epitope antibodies (Table 10).
  • EDE1 C10 binds to ZIKV and DENVs1-4
  • EDE1 C8 binds to ZIKV and DENVs1-4
  • EDE2 A11 binds to DENVs1-4
  • D3-5J7 binds DENV3
  • Z-195 binds ZIKV
  • FLE Z6 is a fusion loop binding, enhancing antibody
  • Further ZIKV and DENV antigens were screened for expression using conformational and linear epitope antibodies (Table 11A-11D).
  • DENV3 and DENV4 designs were tested with other cross-reactive antibodies (e.g., EDE1 C8, EDE2 B7, 5JZ DV3, 5H2 DV4, and FLE Z6).
  • EDE1 C8 EDE1 C8
  • EDE2 B7 EDE2 B7
  • 5JZ DV3, 5H2 DV4, and FLE Z6 cross-reactive antibodies
  • D1-V2, D2-V2, D3-V2, and D4-V2 were selected for continued screening.
  • Each design showed expression levels above that of wild-type and earlier version of the antigens, in addition to having less reactivity to the fusion loop antibodies (FLE 4G2 and FLE Z6).
  • Table 11A Table 11A.
  • DENV1 and DENV2 Relative Expression Data Note: EDE1 C10, binds to ZIKV and DENVs1-4; EDE2 A11, binds to DENVs1-4; D3- 5J7, binds DENV3; sIgN-3C, binds to ZIKV and DENVs1-4; DV1EDI_1F4, DENV1-specific and FLE Z6 is a fusion loop binding, enhancing antibody Table 11B.
  • DENV3 and DENV4 Relative Expression Data Note: EDE1 C8, specific for DENVs1-4 and ZIKV; EDE2 B7, specific for DENVs1-4; DV3 5J7, DENV3-specific; DV45H2, DENV4-specific; and FLE Z6 is a fusion loop binding, enhancing antibody Table 11C.
  • DENV1-4 Relative Expression Data (Broadly Neutralizing Antibodies) Note: EDE1 C8, specific for DENVs1-4 and ZIKV; EDE1 C10, binds to ZIKV and DENVs1-4; EDE2 A11, binds to DENVs1-4; EDE2 B7, specific for DENVs1-4; sIgN-3C, binds to ZIKV and DENVs1-4; and MZ4, specific for ZIKV, DENV2, DENV3 Table 11D.
  • EDE1 C8 specific for DENVs1-4 and ZIKV
  • EDE1 C10 binds to ZIKV and DENVs1-4
  • EDE2 A11 binds to DENVs1-4
  • EDE2 B7 specific for DENVs1-4
  • sIgN-3C binds to ZIKV and DENVs1-4
  • MZ4 specific for ZIKV, DENV2, DENV3 Table 11D.
  • DENV1-4 Relative Expression Data (Serotype-Specific Antibodies; Anti- Fusion Loop Antibodies) Note: 1F4 DV1, DENV1-specific antibody; 2J7 DV3, DENV3-specific antibody; 5H2 DV4, DENV4-specific antibody; FLE 4G2, anti-fusion loop antibody; FLE Z6, anti-fusion loop antibody
  • MS Mass spectrometry
  • the top panel shows the comparison of intracellular and surface proteins in designed and WT antigens (FIG.4A).
  • Cells expressing these VLPs were pelleted down and washed with VLPs before MS inspection of digested peptides. In each case, except for DENV2, the design antigens showed higher overall expression.
  • FIG.4B shows the expression of soluble fractions (VLPs), and in each case, a higher abundance of designed antigens was observed.
  • the wild-type proteins are as follows: WG D1 is DENV1 WT pr/ME; WT D2 is DENV2 WT pr/ME; WT 3 is DENV3 WT pr/ME; WT D4 is DENV4 WT pr/ME, and WT D1-D4 is a co-transfection of the four wild-type proteins (each at 1 ⁇ 4 the amount used for the individual transfections).
  • the four antigens tested were as follows: AS D1 is DV1_008_E/M; AS D2 is DV2-029_E/M; AS D3 is DENV3_HA_60_E/M; AS D4 is DV4-Ind_005_EM; and AS D1-4 is a co-transfection of all four proteins (each at 1 ⁇ 4 the amount used for the individual transfections).
  • the four selected antigens were then screened using negative stain electron microscopy (NSEM). NSEM was used to verify the presence of VLPs in a partially purified sample. In each case, VLPs corresponding to a size of ⁇ 50 ⁇ were observed.
  • VLPs appear to have a smooth appearance indicating prefusion state of the proteins on surface (FIG.5; boxes show the VLPs).
  • the four selected antigens were chosen for purification and further protein analysis. SDS-PAGE was used to validate the designed antigens.
  • FIG.6 demonstrates the results of these candidates, which were expressed in Expi293 cells then purified as described in “Methods” below. Further purification was then performed using streptavidin-tagged EDE1 Fab C8 that bound to the VLPs. The antibody-VLP complexes were captured by a streptavidin column, and VLPs were then eluted.
  • FIG.6 demonstrates the presence of intact DENV E/M heterotetramers at ⁇ 122 kDa in non-reducing conditions (left column for each antigen) and heterodimers at ⁇ 61 kDa in reducing conditions (right column for each antigen), verifying the presence of DENV E/M heterotetrameric complexes containing the engineered disulfide bond and indicating that the protein is properly folded.
  • the selected DENV antigens were screened for expression using conformational and linear epitope antibodies (Table 12). Expression was measured by dot-blot analysis as described above. No binding was observed for the EDE1 GL C10 and DED1 GL C8 antibodies. In each instance, the mature EDE1 and EDE2 monoclonal antibodies were able to detect the designed antigens with higher affinity over their WT counterparts. Only germline EDE2 A11 showed specific signal of binding for the DENV1, -2 and -3 antigens and was unable to bind the DENV4 antigen. EDE1 C8 and C10 didn’t show specific binding signal to the WT or engineered V2 VLPs.
  • WT VLPs were used: DENV1 wild type prM/E VLP, DENV2 wild type prM/E VLP, DENV3 wild type prM/E VLP, and DENV4 wild type prM/E VLP.
  • “All 4” indicates the tetravalent co-transfection
  • “Mock” represents untransfected cells.
  • the designed antigens are indicated as “Stab” and are shown in light gray in FIGs.7A-7D.
  • the designed antigens used were: DV1_008_E/M, DV2-029_E/M, DENV3_HA_60_E/M, and DV4-Ind_005_EM.
  • the cells were pelleted and washed using PBS.
  • FIG.7A serotype specific monoclonal antibodies, 1F4, 2D22, 5J7, and 5H2
  • anti-fusion loop monoclonal antibodies 24211, 24220, and Z6
  • FIG. 7B broadly neutralizing antibodies, A11, B7, C8, C10 and SlgN3C
  • FIG.7D germline versions of the monoclonal antibodies
  • the designed antigens gave higher signals than the WT, except for the undesired anti-fusion loop monoclonal antibodies (FIG.7B).
  • DOT-BLOT Analysis Various monoclonal antibodies (mAbs) were used to assess the expression of engineered VLPs in mammalian cells. Plasmids containing engineered VLPs were transfected into 1 mL Expi293 cells, and cell supernatant was harvested 3 days post-transfection. Cell supernatant was then filtered using a 0.1 ⁇ m filter.1.5 ⁇ L of the filtered (“clarified”) cell supernatant was applied to nitrocellulose membrane and probed using primary Dengue/Zika- or Flavavirus- specific antibodies of human or mouse origin. An anti-human or anti-mouse secondary antibody conjugated to a fluorescent tag was used for final detection, and readout was observed using a ChemiDoc imaging system.
  • Negative Stain Electron Microscopy Engineered VLP candidates showing desired expression were selected for large scale production and structural studies using negative stain electron microscopy. Briefly, 200-500 mL Expi-293 cells were transfected, and cell supernatant was harvested 3 days post-transfection. Cell supernatant was mixed with polyethylene glycol 8000 (PEG8000) and centrifuged to concentrate and pellet VLPs; excess liquid was removed; and the pellet was then resuspended in 50 mM Tris (pH 8.0), 400 mM NaCl and 0.1 mM EDTA. The resuspended pellets were filtered through a 0.45 ⁇ m filter.
  • PEG8000 polyethylene glycol 8000
  • the filtered suspension was further purified using a sucrose gradient of 5%, 10%, 15%, 20% and 25%, and the presence of VLPs was analyzed using a DOT-BLOT assay (see above). Fractions showing the presence of VLPs were pooled and further concentrated using 20% sucrose cushion centrifugation. Pelleted VLPs were resuspended in 500 ⁇ L of 50 mM Tris (pH 8.0), 400 mM NaCl. The resuspended VLPs were then passed through a CaptoCore 700 column (Cytiva) for clarification. The VLPs in the flowthrough were used for negative stain electron microscopy studies.
  • Example 2 In Vitro Analysis of Designed Antigens Antibody binding was tested using Fluorescence Activated Cell Sorting (FACS). Briefly, cells were transfected with the WT or designed antigens alone or in a tetravalent combination (all four DENV serotypes).
  • WT VLPs were used: DENV1 wild type prM/E VLP, DENV2 wild type prM/E VLP, DENV3 wild type prM/E VLP, and DENV4 wild type prM/E VLP.
  • “All 4” indicates the tetravalent co-transfection
  • “Mock” represents untransfected cells.
  • the designed antigens are indicated as “Stab” and are shown in light gray in FIGs.7A-7D.
  • the designed antigens used were: DV1_008_E/M, DV2-029_E/M, DENV3_HA_60_E/M, and DV4-Ind_005_EM.
  • the cells were pelleted and washed using PBS.
  • FIG.7A serotype specific monoclonal antibodies, 1F4, 2D22, 5J7, and 5H2
  • anti-fusion loop monoclonal antibodies 24211, 24220, and Z6
  • FIG.7B broadly neutralizing antibodies, A11, B7, C8, C10 and SlgN3C
  • FIG. 7D germline versions of the monoclonal antibodies
  • the designed antigens gave higher signals than the WT, except for the undesired anti-fusion loop monoclonal antibodies (FIG.7B).
  • Example 3 In Vivo Analysis of Selected Antigens
  • the immunogenicity of selected Dengue virus (DENV) designed antigens was tested in vivo. Briefly, BALB/c mice were administered a prime dose of the selected composition on day 1 and a boost dose of the same composition of day 22. On days 21 and 57, serum samples were taken and analyzed. The monovalent DENV1, DENV2, DENV3, and DENV4 compositions were tested at two total dose levels: 1 ⁇ g and 4 ⁇ g. The compositions tested are shown in Table 13 below. Table 13 – Experimental Groups * for the tetravalent compositions, a 1:1:1:1 ratio was used Using the serum samples collected, neutralization titers (NT 50 ) were measured.
  • NT 50 neutralization titers
  • Monovalent homologous antibody responses were determined and demonstrated that the designed antigens induce serotype-specific antibody responses to a significantly higher level than the control (PBS) (FIG.8).
  • the DENV1 and DENV3 designs induced similar amounts of type-specific antibodies compared to their respective wild-type prME proteins; the DENV2 design induces more type-specific antibodies that its wild-type prME protein; and the DENV4 design induced less type-specific antibodies than its wild-type prME protein.
  • cross-reactivity was examined for each serotype. As is shown in FIG.9A, the engineered DENV1 designs induce cross-reactive antibody responses, and have a different pattern of cross-reactivity than the native VLPs. The results are represented in Table 14 below.
  • Table 14 – Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
  • DENV2 the results are depicted in FIG.9B and demonstrate that the engineered DENV2 designs induce cross-reactive antibody responses, although they induce fewer cross- reactive antibodies than the native VLPs.
  • the results are represented in Table 15 below.
  • Table 15 Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
  • FIG.9C the results are depicted in FIG.9C and demonstrate that the engineered DENV3 designs induce cross-reactive antibody responses, although they induce fewer cross- reactive antibodies than the native VLPs.
  • Table 16 The results are represented in Table 16 below.
  • Table 16 Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
  • DENV4 the results are depicted in FIG.9D and demonstrate that the engineered DENV4 designs induce cross-reactive antibody responses, although they, as well as the wild- type prME, induce the fewest cross-reactive antibodies of all the serotypes tested.
  • the results are represented in Table 17 below.

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Abstract

Provided herein are engineered flavivirus heterotetrameric proteins (.e.g., Envelope and/or Membrane proteins) comprising at least one stabilizing amino acid substitution. The engineered flavivirus proteins form a stabilized heterotetramer structure. Messenger ribonucleic acid vaccines encoding the engineered flavivirus proteins are also provided. The disclosure also provides methods of eliciting an immune response and/or neutralizing antibodies to flavivirus in a subject.

Description

STABILIZED FLAVIVIRUS VACCINES RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63/507,050, filed June 8, 2023, and U.S. Provisional Application No. 63/581,588, filed September 8, 2023, the contents of each of which are incorporated by reference herein in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (M137870275WO00-SEQ-JXV.xml; Size: 524,780 bytes; and Date of Creation: June 7, 2024) are incorporated by reference herein in their entirety. BACKGROUND Flavivirus is a genus of positive-strand RNA viruses in the family Flaviviridae. The genus includes the West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus, Japanese encephalitis virus, Powassan virus, St. Louis Encephalitis virus, Usutu virus, and Spondweni virus which may cause disease (e.g., encephalitis) in humans. Dengue virus is a mosquito-borne flavivirus that causes dengue fever, a severe flu-like illness. There are four distinct, but closely related, serotypes of the virus that cause dengue (DENV-1, DENV-2, DENV-3, and DENV-4). Dengue virus is transmitted to humans through the bites of infected female mosquitoes, primarily the Aedes aegypti mosquito. This species prefers to bite humans and is most active during daylight hours. After virus incubation for about a week, an infected human may develop dengue fever. Dengue fever is characterized by high fever, severe headache, pain behind the eyes, joint and muscle pain, rash, and mild bleeding (such as nose or gum bleed, or easy bruising). In severe cases, it can evolve into severe dengue or dengue hemorrhagic fever, which can cause bleeding, blood plasma leakage, and low platelet count. The disease is endemic in more than 100 countries in the World Health Organization's (WHO) regions of Africa, the Americas, the Eastern Mediterranean, Southeast Asia, and the Western Pacific. Currently, there is only one available vaccine for the prevention of dengue disease caused by dengue virus serotypes 1, 2, 3 and 4, called DENGVAXIA®, which is a live, tetravalent vaccine; however, this vaccine is approved by the FDA only for use in individuals 9 through 16 years of age with laboratory-confirmed previous dengue infection and living in endemic areas. SUMMARY Flaviviruses are pathogens that cause severe morbidity and mortality in humans. These viruses cycle between the arthropod (mosquito or tick) and vertebrate hosts. A few of the most dangerous flaviviruses include Dengue viruses (DENVs), Zika virus (ZIKV), West Nile virus (WNV), Yellow fever virus (YFV), Japanese encephalitis virus, Tick borne encephalitis virus (TBEV), St. Louis Encephalitis virus (SLEV) and emerging viruses like Usutu Virus (USV), Powassan virus (POWV) and Spondweni virus (SPOV) (Pierson et al., Nat Microbiol, 5, 796- 812, 2020)). Flaviviruses are structurally similar to one another (see, e.g., FIG.1A, which illustrates structural similarities between DENV1, DENV2, DENV3, DENV4, and ZIKV). Four dengue virus serotypes (DENV1, 2, 3, and 4) and ZIKV are serologically related viruses that infect humans. The envelope (E) protein dimer in its prefusion conformation is a metastable protein that transforms into the stable post-fusion trimeric form upon exposure to an acidic pH. DENVs have evolved to produce immature particles as decoy antigens that have the outer shell made of (M/E) heterodimers arranged as trimeric spikes with a viral chaperone protein, pr, on the tip of E protein and covalently linked to M protein thus forming 60 (prM/E)3 spikes on the surface. This immature conformation is immunodominant and produces an undesired immune response (Rey et al., Curr Opin Virol., 2018 Jun:24:132-139). Thus, described herein are engineered non-infectious Virus-Like Particles (VLPs) that present as a lattice of heterotetramers formed from E and M proteins. The engineered heterotetramers are stabilized in a native conformation in order to elicit an efficacious protective immune response. The use of such heterotetramers is unique. Previous vaccine designs have relied on structures that do not closely approximate the three-dimensional structure of native flaviviruses and, therefore, are not optimal immunogens. The disclosure, in some aspects, provides a stabilized flavivirus heterotetramer, the heterotetramer comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein the E protein comprises an amino acid substitution at each of two positions, wherein the two positions correspond with position 76 and position 107 of SEQ ID NO: 276, and the M protein comprises a N-terminus and a C-terminus; wherein the N-terminus comprises an N-terminal region comprising at least one amino acid substitution relative to a corresponding wild-type flavivirus M protein. The shared confirmational elements of the flaviviruses allow one of skill in the art to determine those amino acid positions from the related viruses, which correspond to the reference amino acid positions in the Dengue 2 amino acid sequence (see FIG. 1A). Those amino acid positions are amenable to mutation in order to achieve the same stabilizing effect and still allow for the generation of antibodies that will recognize the native flavivirus and, therefore, be protective. It will be understood that in the subsequent paragraphs, the recitation of amino acid positions (e.g., 107 and 76) embraces the corresponding amino acid positions in related flaviviruses which correspond to those positions. In some embodiments, the amino acid substitution at position 107 comprises a substitution with a charged bulky residue. In some embodiments, the amino acid substitution at position 76 comprises a substitution with a charged bulky residue. In some embodiments, the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R). In some embodiments, the at least one substitution within the N-terminal region is selected from the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105; wherein the positions correspond with positions 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105 of SEQ ID NO: 275. In some embodiments, the at least one substitution within the N- terminal region is selected from the following substitutions: (a) the amino acid substitution at position 90 is valine (V) or isoleucine (I); (b) the amino acid substitution at position 91 is isoleucine (I) or cysteine (C); (c) the amino acid substitution at position 93 is isoleucine (I), phenylalanine (F), tyrosine (Y), or tryptophan (W); (d) the amino acid substitution at position 94 is isoleucine (I), leucine (L), asparagine (N), or lysine (K); (e) the amino acid substitution at position 96 is cysteine (C); (f) the amino acid substitution at position 97 is isoleucine (I) or cysteine (C); (g) the amino acid substitution at position 99 is isoleucine (I) or leucine (L); (h) the amino acid substitution at position 101 is methionine (M), isoleucine (I), or phenylalanine (F); (i) the amino acid substitution at position 103 is glycine (G), alanine (A), serine (S), or cysteine (C); (j) the amino acid substitution at position 104 is serine (S); and (k) the amino acid substitution at position 105 is cysteine (C); wherein the positions correspond with positions 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105 of SEQ ID NO: 275. In some embodiments, the E protein comprises an amino acid substitution at each of two positions, wherein the two positions correspond with position 76 and position 107 of SEQ ID NO: 276, and the E protein comprises the wild-type amino acid at position 106, wherein the position corresponds with position 106 of SEQ ID NO: 276. In some embodiments, the E protein further comprises at least one additional substitution selected from the group consisting of: an amino acid substitution at position 131; an amino acid substitution at position 194; and an amino acid substitution at position 134; wherein the positions correspond with positions 131, 194, and 134 of SEQ ID NO: 276. In some embodiments, the at least one additional substitution is selected from the following amino acid substitutions: (a) the amino acid substitution at position 131 is alanine (A), asparagine (N), or leucine (L); (b) the amino acid substitution at position 194 is aspartic acid (D); and (c) the amino acid substitution at position 134 was asparagine (N) or aspartic acid (D); wherein the positions correspond with positions 131, 194, and 134 of SEQ ID NO: 276. In some embodiments, the E protein and the M protein comprise a fusion protein, the fusion protein comprising, from N-terminus to C-terminus, the M protein and the E protein. In some embodiments, the E protein and the M protein comprise a fusion protein, the fusion protein comprising, from N-terminus to C-terminus, the E protein and the M protein and optionally wherein a linker is positioned between the E protein and the M protein, further optionally wherein the linker is a flexible linker. In some embodiments, the linker comprises GGGG (SEQ ID NO: 364) or GGPG (SEQ ID NO: 365). In some embodiments, the E protein further comprises an amino acid substitution at position 495, wherein the position corresponds with position 495 of SEQ ID NO: 276; and/or the M protein further comprises an amino acid substitution at position 90, wherein the position corresponds with position 90 of SEQ ID NO: 275. In some embodiments, the amino acid substitution of the E protein is a glycine (G) at position 495, wherein the position corresponds with position 495 of SEQ ID NO: 276; and/or wherein the amino acid substitution of the M protein is a glycine (G) at position 90, wherein the position corresponds with position 90 of SEQ ID NO: 275. In some embodiments, the heterotetramer further comprises a cavity created by: amino acid substitutions at positions 262 and 265 of the E protein, wherein the positions correspond with positions 262 and 265 of SEQ ID NO: 276; and amino acid substitutions at position 97 of the M protein, wherein the position corresponds with position 97 of SEQ ID NO: 275. In some embodiments, the amino acid substitutions of positions 262 and 265 of the E protein are each an alanine (A) or serine (S), wherein the positions correspond to positions262 and 265 if SEQ ID NO: 276; and the amino acid substitution of position 97 of the M protein is alanine (A), wherein the position corresponds to position 97 of SEQ ID NO: 275. In some embodiments, the E protein further comprises the following amino acid substitutions: a tyrosine (Y), glutamine (Q), or asparagine (N) at position 267; and a lysine (K) or arginine (R) at position 445; wherein the positions correspond to position 267 and 445 of SEQ ID NO: 276. In some embodiments, the N-terminal region of the M protein comprises at least one amino acid substitution of a non-cysteine amino acid with a cysteine amino acid, relative to the corresponding wild-type flavivirus M protein. In some embodiments, the heterotetramer further comprises a signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence selected from SEQ ID NOs: 271 or 272. In some embodiments, the flavivirus is selected from the group consisting of: Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus and Omsk virus. In some embodiments, the flavivirus is Dengue virus. In some embodiments, the flavivirus is Zika virus. In some embodiments, the Zika virus E protein further comprises an introduced glycosylation site. In some embodiments, the introduced glycosylation site comprises an N-X-S motif in positions 67-69, wherein the positions correspond to positions 67-69 of SEQ ID NO: 282. In one embodiment, the disclosure pertains to a nucleic acid molecule encoding a stabilized heterotetramer comprising an E protein and M protein of a flavivirus as described herein. The disclosure, in some aspects and embodiments, provides a DNA plasmid comprising a nucleic acid encoding the E protein and M protein capable of forming any one of the stabilized flavivirus heterotetramers described herein. The disclosure, in some aspects and embodiments, provides a messenger ribonucleic acid (mRNA) vaccine comprising an mRNA polynucleotide having an open reading frame (ORF) encoding a single chain protein comprising the E protein and M protein capable of forming any one of the stabilized flavivirus heterotetramers described herein. In some embodiments, the mRNA vaccine comprises: (A) a first mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NOs: 112, 217, 335-340, and 353-354; (B) a second mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 27, 195, 302-319, 328-334, and 358-360; (C) a third mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 117, 175, 320-327, 341-343, and 361-363; and (D) a fourth mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 122, 229, 344-346, and 355-357. In some embodiments, the mRNA further comprises: (E) a fifth mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 108, 140-142, 177, 285-301, and 347-352. In some embodiments, the mRNA is formulated in a lipid nanoparticle. In some aspects and embodiments, the disclosure provides a composition comprising at least one of the mRNA described herein and a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises 40-55 mol% of a lipid of Compound 1 or A7, 30-45 mol% cholesterol, 5-15 mol% 1,2 distearoyl-sn-glycero-3- phosphocholine (DSPC), and 1-5 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises Compound 1:
Figure imgf000007_0001
In some embodiments, the lipid nanoparticle comprises A7:
Figure imgf000007_0002
In some embodiments, the PEG-modified lipid is PEG2000-DMG. In some embodiments, the mRNA comprises a chemical modification, optionally selected from the group consisting of: pseudouridine (ψ), 1-methyl-pseudouridine (m1ψ), 1-ethylpseudouridine (e1ψ), 5- methoxyuridine (mo5U), 5-methylcytidine (m5C), 5-methoxymethyluridine, 5- methylthiouridine, 1-methoxymethylpseudouridine, 5-methoxycytidine, and any combination thereof. In some embodiments, the mRNA comprises 1-methyl-pseudouridine. In some embodiments, the ORF comprises 1-methyl-pseudouridine, adenosine, guanosine, and cytosine. In some aspects and embodiments, the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject one or more doses of the any one of the stabilized flavivirus heterotetramers described herein in protein or nucleic acid form, e.g., any one of the DNA, e.g., plasmid DNA described herein, any one of the mRNA described herein, or any one of the compositions described herein in an effective amount to produce an immune response. In some aspects and embodiments, the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject one or more doses of the any one of the stabilized flavivirus heterotetramers described herein, any one of the DNA, e.g., plasmid DNA described herein, any one of the mRNA described herein, or any one of the compositions described herein in an amount effective at inducing in the subject a population of neutralizing antibodies that cross reacts with a naturally occurring flavivirus. In some embodiments, the mRNA vaccine is administered intramuscularly. In some embodiments, the flavivirus is Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus, Omsk virus, or any combination thereof. In some embodiments, the flavivirus is Dengue virus. In some embodiments, the flavivirus is Zika virus. The disclosure, in some aspects, provides a method of inducing neutralizing antibodies in a subject by administering a stabilized flavivirus heterotetramer comprising: means for inducing an immune response to the flavivirus in the subject; and a flavivirus envelope (E) protein, wherein the E protein comprises an amino acid substitution at each of position 76 and at position 107, wherein the positions correspond to SEQ ID NO: 276; and a flavivirus membrane (M) protein, wherein administration of the flavivirus heterotetramer has increased neutralization antibody titers compared to wild-type flavivirus prME protein. BRIEF DESCRIPTION OF THE DRAWINGS FIGs.1A-1C show regions of mutations described herein. FIG.1A is a sequence alignment between four dengue virus serotypes and Zika virus. The top panel shows the sequence alignment between prM proteins (rows 1-2; DENV1, SEQ ID NO: 273 A; DENV2, SEQ ID NO: 275; DENV3, SEQ ID NO: 277; DENV4, SEQ ID NO: 279; ZIKV, SEQ ID NO: 281). The gray arrow indicates the furin cleavage site between pr and M proteins. The lower panel shows the sequence alignment between E proteins from five viruses (rows 3-8; DENV1, SEQ ID NO: 274; DENV2, SEQ ID NO: 276; DENV3, SEQ ID NO: 278; DENV4, SEQ ID NO: 280; ZIKV, SEQ ID NO: 282). Black background highlights the identical sequences among viruses and black solid boxes indicate similar sequence. Dotted boxes indicate the areas mutated in antigens described herein. FIGs.1B and 1C illustrate the structure of a flavivirus (M/E)2 heterotetramer from a virion. The flavivirus E protein ectodomain consists of three domains (EDI, EDII, and EDIII) followed by a transmembrane region labelled as “stem” in the figure. The E protein interacts with the transmembrane protein (M) that provides a scaffold and curvature to the E protein dimer. FIG.1B shows the top view of (M/E)2 heterotetramer domain (top panel). The M protein and the stem region provide a scaffold for the E protein homodimer ectodomain region as shown in the side view (bottom panel). Stabilizing mutations are indicated by arrows. FIG.1C provides an illustration of the stem and M protein scaffold and E dimer (top panel) and an open book view of the same (bottom panel). The antigens described herein are unique because they comprise heterotetramer structures, which stabilize the antigen in a prefusion conformation and lead to a stronger immune response than E-dimers or wild-type proteins. FIG.2 shows electron microscopy analysis of virus-like particles (VLPs) formed using engineered proteins described herein. Negative stain electron microscopy and cryogenic (cryo- EM) imaging were used. VLPs imaged included DENV152 (DENV1_52_E/M), DENV2102 (DENV2-M/E_102,) DENV354 (DENV3_54_E/M VLP) and ZIKV 50 (ZIKV_50_E/M). DENV2 102 + Fab C8 indicates DENV2-M/E_102 VLPs in complex with Fab fragments of bnAb C8. ZIKV 50 + Fab C8 indicates ZIKV_50_E/M VLPs in complex with Fab fragments of bnAb C8. The box in each panel highlights the VLPs. VLPs of sizes 40 to 60 nm were observed for each construct. FIG.3 shows an SDS-PAGE gel of ZIKV_HA_50_E/M_VLPs following purification, demonstrating the presence of covalent (EM)2 heterotetramer VLPs. Expi293 cells were transfected using plasmid containing a ZIKV_HA_50_E/M gene. The supernatant was harvested after 72 hours and the VLPs were purified using sucrose gradient method as described previously (Shen et al., eLife 7:e38970). These partially purified VLPs were further purified by affinity chromatography using Fab C8 as bait. Purified samples were run on SDS-PAGE in reducing (+ βME) and non-reducing conditions (- βME). M; molecular weight marker. In this E/M design, E and M proteins are a single chain and the engineered (EM)2 heterotetramer is covalently stabilized and thus in non-reducing conditions this protein runs at a molecular weight of ~130 kDa. The Fab C8 band can be seen at approximately 50 kDa, which also contains the covalently linked heavy and light chains. Under reducing conditions, the EM single chain protein was observed at a molecular weight of approximately 65 kDa (M protein ~ 8.5 kDa + E protein ~ 55 kDa). Fab C8 was also reduced into heavy chain (HC) and light chain (LC) bands. The first two lanes show Fab C8 in non-reducing and reducing conditions, respectively. In reducing conditions (+ βME), the Fab C8 heavy chain (HC) and light chain (LC) are separated. M; marker. FIGs.4A-4B show the relative abundance of intracellular and surface proteins (FIG.4A) and of soluble fractions (VLPs) (FIG.4B) following monovalent transfection (left graphs) and following tetravalent transfection (right graphs). AS indicates exemplary designed proteins (V1 candidates) and WT indicates wild type proteins of 4 exemplary Dengue viruses. Briefly, 72 hours post-transfection in 24 well plate, 100 ul of cell suspension was taken out and pelleted using by centrifugation at 500g for 5 min. In FIG.4A, pelleted cells were washed with PBS and subjected to mass-spectrometry evaluation for quantifying the relative expression of designs vs WT. High relative abundance for designs was observed compared to the WT. FIG.4B shows the supernatant tested for secreted VLP/ protein presence using mass-spectrometry after centrifugation. The relative peptide count between the design and WT protein was determined, for e.g. in monovalent transfection, there was approximately a 40000 higher peptide count for AS D1 compared to the WT D1. FIG.5 shows electron microscopy analysis of (ME)2 stabilized VLPs engineered VLPs using negative stain electron microscopy and cryogenic (cryo-EM) imaging. VLPs imaged included DV1-V2, DV2-V2, DV3-V2, and DV4-V2. The box in each panel highlights the VLPs. VLPs of sizes 40 to 60 nm were observed for each construct. FIG.6 shows an SDS-PAGE gel of DENV VLPs (V2 designs) following purification as described in FIG.3, demonstrating the presence of covalent (EM)2 heterotetramers in Expi203 cells. FIGs.7A-7D show flow cytometry results demonstrating selected DENV antigen (V1 designs) binding to specific antibodies. Each antigen, designed (denoted as “Stab”, right bar) or wild-type (WT, left bar), was tested individually (DENV1, DENV2, DENV3, DENV4) and together (“All 4”). The percent of positive cells (top row) and the mean fluorescence intensity (bottom row) are shown. Cell samples were prepared as described in FIG.4A. A11; mAb EDE2 A11, G-A11; a germline version of the EDE2 mAb A11, B7; mAb EDE2 B7, C8, mAb EDE1 C8, C10; mAb EDE1 C10, G-C10; a germline version of the EDE1 mAb C10, and mAb SlgN3C are the bnAbs that cross-react with all four DENVs to varying extent. D1-F4; mAb 1F4 (specific to DENV1), 2D22; mAb 2D22 (specific to DENV2), 5J7; mAb 5J7 (specific to DENV3), and 5H2; mAb 5H2 (specific to DENV4).24211; mAb ADI-24211, 24220; mAb ADI-24211 and Z6; mAb Z6 are anti-fusion loop antibodies that cross-react with all four DENVs. FIG.8 is four graphs showing monovalent homologous antibody responses (neutralization titers) in vivo. Briefly, BALB/c mice were immunized with the LNPs containing mRNA that encoded for wild type (WT) DENVs1-4 VLPs or the (ME)2 stabilized VLPs (V1). PBS; no LNPs were injected - sterile PBS used instead, WT 1; 1 ug of LNPs encoding WT DENV VLP were injected. V11; 1 ug of the V1 encoding LNPs were injected. V14; 4 ug of the V1 encoding LNPs were injected. A prime (day 1) boost (day 22) regime was followed and blood samples were collected from mice on day 57. Sera from the collected blood samples were used for neutralization experiments and the results of neutralization (NT50) are shown here. The table below the NT50 bars shows the strains used in immunization and neutralization assays. FIGs.9A-9D show cross-reactive antibody responses (neutralization titers) following DENV1 vaccination (FIG.9A), DENV2 vaccination (FIG.9B), DENV3 vaccination (FIG.9C), and DENV4 vaccination (FIG.9D). Control is WT samples. FIG.10 is four graphs showing serotype-specific responses following administration of the tetravalent composition (measured by neutralization titer). FIG.11 shows the immunological balance across the four DENV serotypes at the 1 µg (left graph) and 4 µg (right graph) doses. DETAILED DESCRIPTION Flaviviruses are icosahedral structures having a lipid envelope covered densely with surface projections composed of multiple copies of M (membrane) and multiple copies of E (envelope) glycoproteins. The E glycoproteins are organized as dimers, paired horizontally head to tail, on the virion surface. Flaviviruses are approximately 50 nm spherical particles that, in their mature, infectious form have an outer shell made of 90 membrane (M) protein and envelope (E) protein heterotetramers (M/E)2 arranged in a herringbone pattern. This (M/E)2 outer shell is the main target of the neutralizing antibody response and cross-reactivity among flaviviruses has been reported as the M and E proteins are not only structurally conserved among these viruses but can also have a high sequence identity of > 50% (Rey et al., Cell, 172(6), 1319- 1334, 2018; Duquerroy et al., Encyclopedia of Virol.1, 290-302, 2021) (FIG.1A). While the immune response against the prefusion (M/E)2 heteroterameric form is protective in nature, the response against immature form is often non-neutralizing and can lead to antibody-dependent enhancement (ADE) of the disease. Protective antibody responses during natural infection mostly comprise serotype-specific antibodies, and very rarely by broadly neutralizing antibodies (bnAbs). These protective antibodies target the mature shell of virus particles and lock the outer shell in a pre-fusion conformation. The only known class of bnAbs that targets all four DENVs and ZIKV is called anti-E dimer epitope antibodies (Fey et al., EMBO Rep.2018 Feb; 19(2):206-224; Dejnirattisai et al., Nature Immunology, 16, 170-177; Sharma et al., Cell.2021 Dec 9;184(25):6052-6066). Previous flavivirus vaccine designs focused on stabilizing the E dimer interface present on the ectodomain or generating soluble E proteins (Rouvinski et al., Nature Commun., 8, 15411, 2017; Slon-Campos et al., Nat Immunol., 20, 1291-1298, 2019; Kudlacek et al., Science Advances, 7(42), 2021; Phan et al., J of Biol Chem., 298(7): 102079, 2022). Such soluble E dimers have a conformation that is different from that seen in naturally occurring flaviviruses. That conformation does not promote the formation of antibodies that are optimal for generating an immune response against flavivirus infections. Thus, described herein are engineered non-infectious Virus-Like Particles (VLPs) that form a lattice of heterotetramers formed from E and M proteins (FIGs.1B and 1C). Because the engineered heterotetramers are stabilized in a native conformation, they will elicit an efficacious protective immune response. The engineered VLPs described herein, are stabilized in a prefusion conformation through a combination of mutations in the E protein and the M protein resulting in covalent and non-covalent interactions. FIG.1A shows a sequence alignment between the four DENV serotypes and ZIKV, where exemplary positions mutated for stabilization are boxed. This sequence alignment is based on the conformational similarity among these viruses and the numbering of positions amenable to mutation in a reference sequence (e.g., Dengue 2) can be used to determine those positions in related sequences that are mutated to achieve the same result. In particular, mutations in the bc loop and fusion loop of the E protein, as well as the N-terminal of the M protein were found to stabilize the E protein in its prefusion conformation. As is described in the Examples, the structure-guided antigen designs were applied to multiple different flavivirus, demonstrating the applicability of the substitutions to flavivirus due to the high structural similarity between the flaviviruses. Stabilized heterotetramers were designed for DENV (all four serotypes) and ZIKV and were then tested for expression and presence of the prefusion conformation heterotetramer. Additionally, engineered VLPs for yellow fever virus (YFV) designed using the structure-guided approach described herein showed improved expression and presence of the prefusion heterotetramer conformation. Stabilized Flavivirus Heterotetramers Described herein are engineered flavivirus antigens comprising at least one amino acid substitution relative to their respective wild-type proteins and which form stabilized heterotetramers. A stabilized flavivirus heterotetramer, as used herein, refers to a complex of four flavivirus proteins, including two E proteins and two M proteins having amino acid substitutions relative to wild-type E and M proteins. The amino acid substitutions stabilize the complex of four proteins in a prefusion heterotetramer conformation. As is shown in the instant Examples, stabilizing the heterotetramer in its prefusion conformation elicits an optimal immune response because the native curvature of the E protein is preserved. In the instant constructs, each of the two E proteins includes at least a substitution at positions 76 and 107 (corresponding to a wild-type DENV2 E protein; SEQ ID NO: 276). Position 76 of the DENV2 E protein is located in the bc loop region of the E protein. The bc loop is a hydrophobic loop found in the second domain of the E protein and is linked to the Fu loop via a disulfide bond. Position 107 of the DENV2 E protein is located in the fusion loop region of the E protein. The fusion loop of the E protein forms the tip of the second domain of the E protein and is required for the low-pH- driven membrane fusion of the viral membrane with a host endosomal membrane. As the flaviviruses are structurally similar to one another, the corresponding positions (e.g., positions 76 and 107 of the wild-type DENV2 E protein) can be applied to any other flavivirus E protein; for example, FIG.1A illustrates the analogous regions of DENV1-4 and ZIKV). One of ordinary skill in the art would recognize that a substitution at position 76 of DENV2 (in FIG.1A) corresponds to a substitution at position 76 of DENV1, DENV3, and DENV4, and position 77 of ZIKV, for example. For each of the amino acid substitutions described below, the numbered positions refer to DENV2 E and M proteins; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity. In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid (e.g., SEQ ID NO: 276) with an amino acid comprising a charged bulky residue. In some embodiments, the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with aspartic acid (D). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with glutamic acid (E). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with asparagine (N). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with glutamine (Q). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with lysine (K). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with tyrosine (Y). In some embodiments, the amino acid substitution at position 76 of the E protein comprises a substitution of the wild-type amino acid with arginine (R). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid (e.g., SEQ ID NO: 276) with an amino acid comprising a charged bulky residue. In some embodiments, the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with aspartic acid (D). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with glutamic acid (E). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with asparagine (N). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with glutamine (Q). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with lysine (K). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with tyrosine (Y). In some embodiments, the amino acid substitution at position 107 of the E protein comprises a substitution of the wild-type amino acid with arginine (R). In some embodiments, the E protein comprises an amino acid substitution at each of positions 76 and 107 and does not comprise a mutation (e.g., amino acid substitution) at position 106 (for example, when the position corresponds to SEQ ID NO: 276). In some embodiments, the E protein comprises an amino acid substitution at each of positions 76 and 107 and a wild- type amino acid at position 106 (for example, when the position corresponds to SEQ ID NO: 276). In some embodiments, the E protein comprises a further amino acid substitution. The further amino acid substitution, in some embodiments, is located at the inter-dimer interface of the resulting VLP (e.g., corresponding to SEQ ID NO: 276). For each of the amino acid substitutions, the numbered positions refer to DENV2 E protein; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity. In some embodiments, the further amino acid substitution comprises an amino acid substitution at position 131. In some embodiments, the amino acid substitution at position 131 is selected from the group consisting of: alanine (A), asparagine (N), and leucine (L). In some embodiments, the amino acid substitution at position 131 is alanine (A). In some embodiments, the amino acid substitution at position 131 is asparagine (N). In some embodiments, the amino acid substitution at position 131 is leucine (L). In some embodiments, the amino acid substitution at position 194 is aspartic acid (D). In some embodiments, the amino acid substitution at position 134 is asparagine (N) or aspartic acid (D). In some embodiments, the amino acid substitution at position 134 is asparagine (N). In some embodiments, the amino acid substitution at position 134 is aspartic acid (D). In some embodiments the E protein comprises an amino acid substitution at position 131, at position 134, and at position 194. The further E protein amino substitutions may comprise, in positions 131, 134, and 194 any of the following combinations: AND, ADD, NND, NDD, LND, or LDD. In some embodiments, the E protein comprises further amino acid substitution in order to stabilize its interactions with its stem region. For each of the amino acid substitutions, the numbered positions refer to DENV2 E protein; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity. In some embodiments, the E protein comprises an amino acid substitution at position 267 and/or 445 corresponding to SEQ ID NO: 276. In some embodiments, the E protein amino acid mutation is a tyrosine (Y), a glutamine (Q), or an asparagine (N) at position 267. In some embodiments, the E protein amino acid mutation is a lysine (K) or an arginine (R) at position 445. In some embodiments, the E protein comprises, in position 267 and 445, any of the following combinations: YK, YR, QK, QR, NK, or NR. Without wishing to be bound by theory, it is thought that increasing hydrophobic interactions between the M protein and the bottom interface of the E dimer serves to stabilize the heterotetramer. This, in some embodiments, is accomplished by introducing at least one amino acid mutation in the N-terminal region of the M protein (e.g., corresponding to SEQ ID NO: 275). In some embodiments, the at least one amino acid substitution within the N-terminal region on the M protein is located within the N-terminal 38 amino acids (e.g., corresponding to SEQ ID NO: 275). For each of the amino acid substitutions, the numbered positions refer to DENV2 M protein; however, as described above, each substitution can be applied to any flavivirus owing to their structural similarity. In some embodiments, the at least one amino substitution within the N-terminal region of the M protein is selected from the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105. In some embodiments, the at least one amino acid substitution within the N-terminal region of the M protein comprises an amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105. In some embodiments, the M protein comprises an amino acid substitution at position 90. In some embodiments, the M protein comprises an amino acid substitution at position 91. In some embodiments, the M protein comprises an amino acid substitution at position 93. In some embodiments, the M protein comprises an amino acid substitution at position 94. In some embodiments, the M protein comprises an amino acid substitution at position 96. In some embodiments, the M protein comprises an amino acid substitution at position 97. In some embodiments, the M protein comprises an amino acid substitution at position 99. In some embodiments, the M protein comprises an amino acid substitution at position 101. In some embodiments, the M protein comprises an amino acid substitution at position 103. In some embodiments, the M protein comprises an amino acid substitution at position 104. In some embodiments, the M protein comprises an amino acid substitution at position 105. In some embodiments, the amino acid substitution at position 90 of the M protein is valine (V). In some embodiments, the amino acid substitution at position 90 of the M protein isoleucine (I). In some embodiments, the amino acid substitution at position 91 of the M protein (e.g., corresponding to SEQ ID NO: 275) is cysteine (C). In some embodiments, the amino acid substitution at position 91 of the M protein isoleucine (I). In some embodiments, the amino acid substitution at position 91 of the M protein (i.e., cysteine) is paired with an amino acid substitution of cysteine (C) at position 262 of the E protein, to form a disulfide bond. In some embodiments, the amino acid substitution at position 93 of the M protein (e.g., corresponding to SEQ ID NO: 275) is phenylalanine (F). In some embodiments, the amino acid substitution at position 93 of the M protein is tyrosine (Y). In some embodiments, the amino acid substitution at position 93 of the M protein is tryptophan (W). In some embodiments, the amino acid substitution at position 94 of the M protein (e.g., corresponding to SEQ ID NO: 275) is isoleucine (I). In some embodiments, the amino acid substitution at position 94 of the M protein is leucine (L). In some embodiments, the amino acid substitution at position 94 of the M protein is asparagine (N). In some embodiments, the amino acid substitution at position 94 of the M protein is lysine (K). In some embodiments, the amino acid substitution at position 96 of the M protein (e.g., corresponding to SEQ ID NO: 275) is cysteine (C). In some embodiments, the amino acid substitution at position 96 of the M protein (i.e., cysteine) is paired with an amino acid substitution of cysteine (C) at position 267 of the E protein, to form a disulfide bond. In some embodiments, the amino acid substitution at position 97 of the M protein (e.g., corresponding to SEQ ID NO: 275) is isoleucine (I). In some embodiments, the amino acid substitution at position 97 of the M protein is cysteine (C). In some embodiments, the amino acid substitution at position 97 of the M protein (i.e., cysteine) is paired with an amino acid substitution of cysteine (C) at position 495 of the E protein, to form a disulfide bond. In some embodiments, the amino acid substitution at position 99 of the M protein (e.g., corresponding to SEQ ID NO: 275) is isoleucine (I). In some embodiments, the amino acid substitution at position 99 of the M protein is leucine (L). In some embodiments, the amino acid substitution at position 101 of the M protein (e.g., corresponding to SEQ ID NO: 275) is methionine (M). In some embodiments, the amino acid substitution at position 101 of the M protein is isoleucine (I). In some embodiments, the amino acid substitution at position 101 of the M protein is phenylalanine (F). In some embodiments, the amino acid substitution at position 103 of the M protein (e.g., corresponding to SEQ ID NO: 275) is glycine (G). In some embodiments, the amino acid substitution at position 103 of the M protein is alanine (A). In some embodiments, the amino acid substitution at position 103 of the M protein is serine (S). In some embodiments, the amino acid substitution at position 103 of the M protein is cysteine (C). In some embodiments, the amino acid substitution at position 103 of the M protein (i.e., cysteine) is paired with an amino acid substitution of cysteine (C) at position 280 of the E protein, to form a disulfide bond. In some embodiments, the amino acid substitution at position 104 of the M protein (e.g., corresponding to SEQ ID NO: 275) is serine (S). In some embodiments, the amino acid substitution at position 105 of the M protein (e.g., corresponding to SEQ ID NO: 275) is cysteine (C). In some embodiments, the amino acid substitution at position 105 of the M protein (i.e., cysteine) is paired with an amino acid substitution of cysteine (C) at position 280 of the E protein, to form a disulfide bond. In some embodiments, the E protein and the M protein comprise a fusion protein. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the M protein and the E protein. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the E protein and the M protein. In some embodiments, a linker is positioned between the E protein and the M protein (i.e., from N-terminus to C-terminus, the fusion protein comprises the E protein, the linker, and the M protein). In some embodiments, the linker is a flexible linker. Without wishing to be bound by theory, it is thought that the inclusion of the flexible linker facilitates assembly of the heterotetramer and VLPs. In some embodiments, the linker comprises, or consists of, GGGG (SEQ ID NO: 364). In some embodiments, the linker comprises, or consists of, GGPG (SEQ ID NO: 365). In some embodiments, further amino acid substitutions are introduced to increase the flexibility of the linker region. In some embodiments, the further amino acid substitutions comprise an amino acid substitution at position 495 of the E protein and an amino acid substitution at position 90 of the M protein. In some embodiments, the further amino acid substitutions comprise a glycine (G) at position 495 of the E protein and a glycine (G) at position 90 of the M protein. In some embodiments, amino acid substitutions were used to generate a cavity between the E protein and the M protein to facilitate entry of the linker. In some embodiments, the cavity is introduced by amino acid substitutions at positions 262 and 265 of the E protein and position 97 of the M protein. In some embodiments, the amino acid substitutions comprise an alanine (A) or a serine (S) at each of position 262 and position 265 of the E protein; and an alanine (A) at position 97 of the M protein. The tables below present a number of different engineered flavivirus fusion proteins. In some embodiments, the antigens for VLPs and the Pr protein sequence is followed by the M protein and then the E protein (“PrM/E”). In some embodiments, the protein comprises the M and E proteins (either M/E or E/M order). In some embodiments, the protein is a single chain (e.g., the mRNA encoding a single chain protein). In some embodiments, stabilizing mutations are included. In some embodiments, when the antigen is in the E/M order, the E protein is connected to the M protein via a linker (e.g., a GS linker), or a Rosetta-based linker. In some embodiments, the flavivirus fusion protein further comprises a Capsid (C) protein and the NS2b/NS3 protease sequences from the corresponding virus (e.g., DENV or ZIKV) (denoted as “C-NS2B3” in the tables below). Capsid and NS2b/NS3 protease are involved in the maturation process of flaviviruses and their inclusion, it is thought, results in a higher yield of the VLPs. In some embodiments, the inclusion of a Capsid protein and NS2b/NS3 protease may result in the symmetric assembly of the E/M or M/E heterotetramers. In some embodiments, the Capsid protein is further mutated to reduce the basic charge (e.g., with an R85M mutation relative to NCBI Reference Sequence: NP_739591.2). In some embodiments, the engineered flavivirus protein comprises amino acid mutations resulting in formation of a glycosylation site. In some embodiments, the glycosylation site is introduced in an engineered ZIKV E protein, for example, using a N67X68S69 motif relative to SEQ ID NO: 282. It is thought that this mutation immune-focuses the response from the ZIKV VLPs to the E-dimer interface, eliciting broadly neutralizing antibodies. Non-limiting examples of DENV and ZIKV and YFV proteins of the present disclosure are provided in Tables 1-4. The signal peptide (e.g., MLRLLLRHHFHCLLLCAVWATPCLA (SEQ ID NO: 271) or MKAILVVLLYTFTTANA (SEQ ID NO: 272)) in any one or more of the proteins of Tables 1-4 may be included, modified, omitted, or exchanged for a different signal peptide. Amino acid substitutions relative to the corresponding wild-type sequence are shown in bold below. In some embodiments, a flavivirus is of a particular strain of flavivirus. In some embodiments, a YFV is an Asibi strain. In some embodiments, a YFV is a 17D strain. In some embodiments, a YFV is Brazilian strain (e.g., YFV2015-2018). In some embodiments, a YFV is a Venezuelan strain (e.g., Venezuela 2000). In some embodiments, a YFV is an African strain (e.g., West Africa I, West Africa II, East Africa, East/Central Africa, or Angola). In some embodiments, YFV is a South American strain (e.g., South America I or South America II). Non-limiting examples of wild-type DENV, ZIKV and YFV sequences may be found at GenBank Accession Nos: QHJ14106.1 (DENV1), QBK46950.1 (DENV2), UJT50354.1 (DENV3), UDW38823.1 (DENV4), AZS35368.1 (ZIKV), AAX47570.1/ NP_041726.1 (YFV) and are listed below. DENV1 E Protein AFHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPQITETEPDD VDCWCNATDTWVTYGTCFQTGEHRRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQRVETWA LRHPGFTVMALFLAHAIGTSITQKGIIFILLMLVTPSMA (SEQ ID NO: 273) DENV1 M Protein MRCVGIGSRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTDPAVLRKLCIEAK ISNTTTDSRCPTQGEATLVEEQDANFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGK IVQYENLKYSVIVTVHTGDQHQVGNESTEHGTTATITPQAPTTEIQLTDYGALTLDCSPRTGLD FNEMVLLTMKEKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQ EGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKELAETQHGTV LVQIKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVIGAGE KALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLVHQIFGTAYGVLF SGVSWTMKIGIGVLLTWLGLNSRSTSLSMTCIAVGLVTLYLGVMVQA (SEQ ID NO: 274) DENV2 E Protein AFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLLRQNEPED IDCWCNSTSTWVTYGTCTTMGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWI LRHPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMT (SEQ ID NO: 275) DENV2 M Protein MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAK LTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGK VVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLD FNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQ EGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTI VIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEP GQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAF SGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA (SEQ ID NO: 276) DENV3 E Protein AFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHITEVEPED IDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWA LRHPGFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMT (SEQ ID NO: 277) DENV3 M Protein MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGK ITNITTDSRCPTQGEATLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGK VVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFN EMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEG AMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILI KVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNA LKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSG VSWVMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGAVVQA (SEQ ID NO: 278) DENV4 E Protein AFHLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPED IDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWI LRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYG (SEQ ID NO: 279) DENV4 M Protein MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEAS ISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFLCSGKITGN LVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGID FNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKAPHAKRQDVTVLGSQ EGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTT VVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPFAENTNSVTNIELEPPFGDSYIVIGVGD SALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMF GGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQA (SEQ ID NO: 280) ZIKV E Protein AAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPD DVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVEN WIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYS (SEQ ID NO: 281) ZIKV M Protein IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEAS ISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGK SIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEP RTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVV VLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAE TLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGD SYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQI FGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA (SEQ ID NO: 282) YFV E Protein: AHCIGITDRDFIEGVHGGTWVSATLEQGKCVTVMAPDKPSLDISLQTVAIDGPAEARKVCYSAV LTHVKINDKCPSTGEAHLAEENDGDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTCAKSMSLF EVDRTKIQYVIRAQLHVGAKQENWNTDIKTLKFDALSGSQEAEFTGYGKATLECQVQTAVDFGN SYIAEMEKDSWIVDRQWAQDLTLPWQSGSGGTWREMHHLVEFEPPHAATIRVLALGNQEGSLKT ALTGAMRVTKDENDNNLYKLHGGHVSCRVKLSALTLKGTSYKMCTDKMSFVKNPTDTGHGTVVM QVKVPKGAPCKIPVIVADDLTAAVNKGILVTVNPIASTNDDEVLIEVNPPFGDSYIIVGTGDSR LTYQWHKEGSSIGKLFTQTMKGAERLAVMGDAAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGG LSWITKVIMGAVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA (SEQ ID NO: 283) YFV M Protein: AIDLPTHENHGLKTRQEKWMTGRMGERQLQKIERWFVRNPFFAVTALTIAYLVGSNMTQRVVIA LLVLAVGPAYS (SEQ ID NO: 284) Table 1. PrM/E based proteins
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Table 2. M/E based VLP proteins
Figure imgf000026_0002
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Table 3. E/M based VLP proteins
Figure imgf000047_0002
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Table 4. M/E eVLP based proteins
Figure imgf000075_0002
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
In some embodiments, a vaccine of the present disclosure comprises a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363. In some embodiments, a vaccine of the present disclosure comprises a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212- 213, 285-301, and 347-352. In some embodiments, a vaccine of the present disclosure comprises a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214. In some embodiments, a vaccine of the present disclosure comprises a nucleic acid comprising an open reading frame encoding a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246- 255, 302-346, and 353-363. In some embodiments, a vaccine of the present disclosure comprises a nucleic acid comprising an open reading frame encoding a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352. In some embodiments, a vaccine of the present disclosure comprises a nucleic acid comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214. In some embodiments, a vaccine of the present disclosure comprises a DNA comprising an open reading frame encoding a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363. In some embodiments, a vaccine of the present disclosure comprises a DNA comprising an open reading frame encoding a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352. In some embodiments, a vaccine of the present disclosure comprises a DNA comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238- 245, 187-188, and 214. In some embodiments, a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a DENV protein, wherein the DENV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246- 255, 302-346, and 353-363. In some embodiments, a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a ZIKV protein, wherein the ZIKV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352. In some embodiments, a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214. In some embodiments, each of the E proteins in the heterotetramer has the same amino acid substitutions as one another. In some embodiments, the two E proteins in the heterotetramer have at least one amino acid substitution different from one another. In some embodiments, each of the M proteins in the heterotetramer has the same amino acid substitution(s) as one another. In some embodiments, the two M proteins in the heterotetramer have at least one amino acid substitution different from one another. The flavivirus proteins of the present disclosure are antigenic, i.e., they promote an immune response when administered to a subject. The subject antigens can be administered as a protein or as a nucleic acid molecule encoding the protein. When delivered to a subject in nucleic acid form, the antigenic flavivirus heterotetramer is expressed by cells in the subject and stimulates the subject’s immune system. When delivered as an mRNA, the mRNA is provided in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles) such that upon administration to cells, tissues or subjects, the mRNA is taken up by cells which, in turn, express the protein(s) encoded by the mRNA. In some embodiments, the vaccines of the present disclosure comprise mRNA encoding the desired DENV antigen(s), which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired antigen(s). In order to facilitate delivery of the mRNA to the cells of the body, the RNA is formulated (e.g., encapsulated) in a lipid nanoparticle. Upon delivery and uptake by cells of the body, the RNA is translated in the cytosol and the antigens are generated by the host cell machinery. The antigens are presented by the host cells and elicit an adaptive humoral and cellular immune response. Neutralizing antibodies are directed against the expressed antigens, and hence the antigens are, in the case of stabilized heterotetramers, relevant target antigens for use as vaccines because they closely mirror the conformation of naturally occurring flaviviruses. mRNA technology is amenable to rapid design and testing of mRNA encoding a variety of antigens. Moreover, rapid production of mRNA coupled with formulation in appropriate delivery vehicles (e.g., lipid nanoparticles), can proceed quickly and can rapidly produce mRNA vaccines at large scale. Potential benefit also arises from the fact that antigens encoded by the mRNAs of the present disclosure are expressed by the cells of the subject, e.g., are expressed by the human body, and thus the subject, e.g., the human body, serves as the “factory” to produce the antigens which, in turn, elicits the desired immune response. The vaccines, as provided herein, may include a single mRNA or multiple RNAs encoding two or more antigens of the same or different flaviviruses (e.g., different strains of a flavivirus). Also provided herein are combination vaccines that include mRNA encoding one or more DENV antigens and one or more antigen(s) of a different virus (e.g., a second flavivirus). Thus, the vaccines of the present disclosure may be combination vaccines that target one or more antigens of the same strain/species, or one or more antigens of different strains/species, e.g., antigens that induce immunity to organisms that are found in the same geographic areas where the risk of flavivirus infection (e.g., DENV) is high or organisms to which an individual is likely to be exposed to when exposed to the flavivirus. Engineered Flavivirus Proteins In some embodiments, the engineered flavivirus proteins comprise additional amino acid substitutions. Examples of such amino acid substitutions are provided below. For clarity, mutations are described using amino acid numbering corresponding to a wild-type sequence (e.g., wild-type DENV2 E protein, SEQ ID NO: 274). The person of ordinary skill in the art will appreciate that mutations disclosed in relation to the wild-type sequence may be applied to other flavivirus protein amino acid sequences. To apply a substitution disclosed with numbering corresponding to the wild-type sequence to an engineered protein, the skilled artisan may align the engineered protein’s amino acid sequence to the amino acid sequence of the wild-type sequence with which the substitution is numbered. For example, to apply an A95G substitution numbered according to SEQ ID NO: 281 (the listed amino acid sequence), to a reference ZIKV M protein, the skilled artisan would align the reference ZIKV M protein amino acid sequence to SEQ ID NO: 281, and introduce a glycine (G) at the residue of the reference ZIKV M protein that corresponds to A95 of SEQ ID NO: 281. The skilled artisan will appreciate the substitutions disclosed in relation to a listed amino acid sequence (e.g., SEQ ID NO: 281) may be referred to in the form X1[#]X2, where X1 is the amino acid at position [#] in a listed amino acid sequence, and X2 is the amino acid introduced by replacement of the X1 at position [#]. For example, a glycine substitution at position 95 may also be referred to as an A95G substitution, when using SEQ ID NO: 281 as a listed amino acid sequence, because the alanine (A) at position 95 of SEQ ID NO: 281 is replaced with a glycine (G). Such a substitution may also be referred to as an “X2 substitution at position [#]”, meaning that a protein comprises an X2 residue at position [#] (numbered by alignment to a listed sequence), regardless of whether the residue that was present at position [#] in the reference amino acid sequence was X1, or a different residue other than X2. The skilled artisan will understand that substitutions disclosed in the form X1[#]X2 may also be applied to a reference amino acid sequence in the form of “X2 substitution at position [#]” that is agnostic to the residue present in the reference amino acid sequence (numbered by alignment to a listed sequence). DENV1 E Protein In some embodiments, the DENV1 E protein may comprise at least one amino acid substitution. In some embodiments, the DENV1 E protein comprises at least one amino acid substitution at one or more positions selected from T76, R93, G106, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to the DENV1 E protein reference sequence (SEQ ID NO: 274). In some embodiments, the DENV1 E protein comprises at least one substitution selected from T76Q, T76D, G106S, G106T, G106N, G106K, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, T268V, A280C, G445K, A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76D, L107R, T262S, T265A, A280C, and G445K, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76D, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and A280C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76D, G106T, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and A280C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76D, G106N, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and A280C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, G106T, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, G106N, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a T76Q substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a glutamine at position 76 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an G106S substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a serine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a G106T substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a threonine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a G106N substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an asparagine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a G106K substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a lysine at position 106 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an L107R substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an arginine at position 107 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a Q131A substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 131 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an N194D substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an aspartic acid at position 194, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an aspartic acid at position 194 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an H209N substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an asparagine at position 209, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an asparagine at position 209 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a T262S substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a serine at position 262, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a serine at position 262 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a T265A substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 265, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 265 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an A267C substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 267, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 267 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a T268A substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 268, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an alanine at position 268 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an A280C substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 280, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 280 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a G445K substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a lysine at position 445, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a lysine at position 445 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises an A495C substitution relative to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 495, where the positions are numbered according to SEQ ID NO: 274. In some embodiments, the DENV1 E protein comprises a cysteine at position 495 relative to a reference DENV1 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 E protein to SEQ ID NO: 274. DENV1 M Protein In some embodiments, the DENV1 M protein may comprise at least one mutation. In some embodiments, the DENV1 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, A97, H99, V100, L102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or A167, relative to the DENV1 M protein reference sequence (SEQ ID NO: 273). In some embodiments, the DENV1 M protein comprises at least one substitution selected from S93G, V94I, H99C, V100A, L104I, T106G, T106C, T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at V100A and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at S93G, V94I, H99C, V100A, L104I, T106G, and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at S93G, V94I, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises substitutions at S93G, A97N, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an S93G substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a glycine at position 93 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a V94I substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an isoleucine at position 94 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an H99C substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a cysteine at position 99 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a V100A substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an alanine at position 100 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an L104I substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises an isoleucine at position 104 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a T106G substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a glycine at position 106, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a glycine at position 106 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a T108C substitution relative to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 273. In some embodiments, the DENV1 M protein comprises a cysteine at position 108 relative to a reference DENV1 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV1 M protein to SEQ ID NO: 273. DENV2 E Protein In some embodiments, the DENV2 E protein may comprise at least one mutation. In some embodiments, the DENV2 E protein comprises at least one substitution at one or more positions selected from T76, R93, G106, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to the DENV2 E protein reference sequence (SEQ ID NO: 276). In some embodiments, the DENV2 E protein comprises at least one substitution selected from T76D, T76N, T76E, T76Y, G106S, G106T, G106N, G106K, L107R, L107K, L107Q, Q131N, Q131A, Q131L, Q131E, E133A, N194E, N194D, E195Q, E195S, H209Y, H209N, H209D, H209K, T262S, T262C, T265A, A267C, T268A, T268I, T268V, T280C, H317N, T319V, G441A, G445K, A495G, A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, L107R, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and T280C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, L107R, T262S, T265A, H317N, T319V, G441A, and G445K, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262C, T265A, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N194E, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262C, T265A, A267C, T268V, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N194E, H209N, T262C, T265A, A267C, T268V, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N194E, H209N, A267C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268I, and T280C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, A267C, T268V, and T280C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, G106S, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, G106T, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, G106N, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76D, G106K, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, G106S, L107R, Q131A, N194D, H209N, T262C, T265A, A267C, T268V, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, G106S, L107R, Q131A, L191I, N194D, H209N, T262S, T265A, A267C, T268I, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76Q, L107R, Q131A, N194D, H209N, T262S, T265A, and A267C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, and T280C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises substitutions at T76E, L107R, Q131A, N194D, H209N, T262S, T265A, A267C, T268A, T280C, and A495C, relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76D substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76E substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T76Y substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 76, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 76 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an G106S substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a G106T substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a threonine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a G106N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a G106K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 106 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an L107R substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an arginine at position 107 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an L107K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 107, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 107 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an L107Q substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 107, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 107 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131A substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131L substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a leucine at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a leucine at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a Q131E substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 131, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 131 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an E133A substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 133, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 133 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an N194E substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 194, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamic acid at position 194 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an N194D substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 194, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 194 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an E195Q substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 195, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glutamine at position 195 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an E195S substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 195, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 195 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209Y substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a tyrosine at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209D substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an aspartic acid at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H209K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 209, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 209 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T262S substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 262, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a serine at position 262 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T262C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 262, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 262 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T265A substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 265, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 265 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an A267C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 267, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 267 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T268A substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 268, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 268 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T268I substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an isoleucine at position 268, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an isoleucine at position 268 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T268V substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a valine at position 268, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a valine at position 268 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T280C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 280, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 280 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an H317N substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 317, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an asparagine at position 317 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a T319V substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a valine at position 319, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a valine at position 319 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a G441A substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 441, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an alanine at position 441 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a G445K substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 445, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a lysine at position 445 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an A495G substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glycine at position 495, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a glycine at position 495 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises an A495C substitution relative to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 495, where the positions are numbered according to SEQ ID NO: 276. In some embodiments, the DENV2 E protein comprises a cysteine at position 495 relative to a reference DENV2 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 E protein to SEQ ID NO: 276. DENV2 M Protein In some embodiments, the DENV2 M protein may comprise at least one mutation. In some embodiments, the DENV2 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to the DENV2 M protein reference sequence (SEQ ID NO: 275). In some embodiments, the DENV2 M protein comprises at least one substitution selected from S93G, V94I, A95C, L96F, L96I, L96W, V97N, H99C, V100A, V100C, V100I, L104H, L104I, E105L, T106G, T106A, T106S, T108C, A117Q, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at V100C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96I, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96W, V97N, H99C, V100I, L104I, E105L, T106A, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96W, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at L96W, H99C, V100I, L104I, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, A95C, H99C, V100C, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, T106S, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V97N, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, T106S, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V97N, H99C, V100C, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises substitutions at S93G, V94I, H99C, V100A, L104I, T106G, and T108C, relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an S93G substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 93 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V94I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 94 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an A95C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 95, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 95 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L96F substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a phenylalanine at position 96, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a phenylalanine at position 96 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L96I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 96, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 96 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L96W substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a tryptophan at position 96, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a tryptophan at position 96 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V97N substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an asparagine at position 97, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an asparagine at position 97 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an H99C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 99 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V100A substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 100 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V100C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 100, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 100 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a V100I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 100, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 100 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L104H substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a histidine at position 104, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a histidine at position 104 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an L104I substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an isoleucine at position 104 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an E105L substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a leucine at position 105, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a leucine at position 105 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T106G substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 106, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glycine at position 106 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T106A substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 106, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an alanine at position 106 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T106S substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a serine at position 106 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a T108C substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a cysteine at position 108 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises an A117Q substitution relative to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glutamine at position 117, where the positions are numbered according to SEQ ID NO: 275. In some embodiments, the DENV2 M protein comprises a glutamine at position 117 relative to a reference DENV2 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV2 M protein to SEQ ID NO: 275. DENV3 E Protein In some embodiments, the DENV3 E protein may comprise at least one mutation. In some embodiments, the DENV3 E protein comprises at least one substitution at one or more positions selected from T76, K93, G106, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A493, relative to the DENV3 E protein reference sequence (SEQ ID NO: 278). In some embodiments, the DENV3 E protein comprises at least one substitution selected from T76Q, T76K, T76E, T76Y, G106S, G106T, G106N, G106K, L107R, L107N, Q131L, Q131A, Q131N, Q131E, E133A, N192D, N192E, E193Q, H207N, H207Y, T260S, T260C, T263A, A265C, T266V, T266A, A278C, A278T, T443K, A493C, A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, L107R, T260S, T263A, T266V, A278C, and T443K, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, and T443K, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, and T443K, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, R208K, T260S, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, R208K, T260C, T263A, A265C, A278T, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Y, L107R, Q131E, E133A, N192E, H207N, R208K, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, L189I, N192D, H207N, T260C, T263A, A265C, T266V, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, G106S, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, G106T, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, G106N, L107R, T260S, T263A, T266V, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, T266A, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76Q, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, T266A, A278C, T443K, and A493G, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises substitutions at T76E, L107R, Q131A, N192D, H207N, T260S, T263A, A265C, T266A, A278C, T443K, and A493C, relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T76Q substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T76K substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 76, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T76E substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 76, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T76Y substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a tyrosine at position 76, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a tyrosine at position 76 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an G106S substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a serine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a G106T substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a G106N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a G106K substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 106 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an L107R substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an arginine at position 107 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an L107N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 107, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 107 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131L substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a leucine at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a leucine at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131A substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a Q131E substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 131, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 131 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an E133A substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 133, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 133 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an N192D substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an aspartic acid at position 192, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an aspartic acid at position 192 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an N192E substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 192, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamic acid at position 192 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an E193Q substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 193, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glutamine at position 193 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an H207N substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 207, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an asparagine at position 207 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an H207Y substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a tyrosine at position 207, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a tyrosine at position 207 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T260S substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a serine at position 260, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a serine at position 260 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T260C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 260, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 260 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T263A substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 263, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 263 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A265C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 265, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 265 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T266V substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a valine at position 266, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a valine at position 266 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T266A substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 266, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an alanine at position 266 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A278C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 278, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 278 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A278T substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 278, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a threonine at position 278 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a T443K substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 443, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a lysine at position 443 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A493C substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 493, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a cysteine at position 493 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises an A493G substitution relative to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glycine at position 493, where the positions are numbered according to SEQ ID NO: 278. In some embodiments, the DENV3 E protein comprises a glycine at position 493 relative to a reference DENV3 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 E protein to SEQ ID NO: 278. DENV3 M Protein In some embodiments, the DENV3 M protein may comprise at least one mutation. In some embodiments, the DENV3 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to the DENV3 M protein reference sequence (SEQ ID NO: 277). In some embodiments, the DENV3 M protein comprises at least one substitution selected from S93G, V94I, A95C, A97V, H99C, V100A, V100C, L104I, T106C, T108C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at V100A and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, V100A, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100C, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, A97V, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100A, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100C, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, and L104I, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, A95C, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, A97V, H99C, V100C, L104I, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, V100A, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100C, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises substitutions at S93G, V94I, H99C, V100A, and T106C, relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an S93G substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a glycine at position 93 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a V94I substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an isoleucine at position 94 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an A95C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 95, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 95 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an A97V substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a valine at position 97, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a valine at position 97 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an H99C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 99 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a V100A substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an alanine at position 100 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a V100C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 100, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 100 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an L104I substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises an isoleucine at position 104 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a T106C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 106, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 106 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a T108C substitution relative to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 277. In some embodiments, the DENV3 M protein comprises a cysteine at position 108 relative to a reference DENV3 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV3 M protein to SEQ ID NO: 277. DENV4 E Protein In some embodiments, the DENV4 E protein may comprise at least one mutation. In some embodiments, the DENV4 E protein comprises at least one substitution at one or more positions selected from T76, R93, G106, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to the DENV4 E protein reference sequence (SEQ ID NO: 280). In some embodiments, the DENV4 E protein comprises at least one substitution selected from T76Q, T76D, T76Y, G106S, G106T, G106N, G106K, L107R, L107E, Q131A, N194E, N194R, E195Q, H209D, H209N, H209Y, A267C, T268A, A280C, T445K, A495C, or A495G, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, L107R, A280C, and T445K, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106T, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106N, L107R, Q131A, H209D, A267C, T268A, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, L107R, Q131A, H209D, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, H209D, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises substitutions at T76Q, G106S, L107R, Q131A, H209D, S262C, A267C, T268I, A280C, and A495C, relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a T76Q substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a glutamine at position 76 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an G106S substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a serine at position 106, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a serine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a G106T substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a threonine at position 106, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a threonine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a G106N substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an asparagine at position 106, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an asparagine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a G106K substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a lysine at position 106, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a lysine at position 106 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an L107R substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an arginine at position 107 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a Q131A substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an alanine at position 131, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an alanine at position 131 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an H209D substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an aspartic acid at position 209, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an aspartic acid at position 209 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an A267C substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 267, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 267 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a T268A substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an alanine at position 268, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an alanine at position 268 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an A280C substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 280, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 280 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a T445K substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a lysine at position 445, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a lysine at position 445 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises an A495C substitution relative to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 495, where the positions are numbered according to SEQ ID NO: 280. In some embodiments, the DENV4 E protein comprises a cysteine at position 495 relative to a reference DENV4 E protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 E protein to SEQ ID NO: 280. DENV4 M Protein In some embodiments, the DENV4 M protein may comprise at least one mutation. In some embodiments, the DENV4 M protein comprises at least one substitution at one or more positions selected from S93, V94, A95, L96, T97, H99, S100, M102, L104, E105, T106, R107, A108, E109, S113, S114, A117, K119, Q122, E125, S126, R130, S165, Y166, or G167, relative to the DENV4 M protein reference sequence (SEQ ID NO: 279). In some embodiments, the DENV4 M protein comprises at least one substitution selected from S93G, V94I, H99C, S100A, S100C, L104I, T106G, A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S100A and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, V94I, S100A, T106G, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, V94I, H99C, S100C, L104I, T106G, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, H99C, S100C, L104I, T106G, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises substitutions at S93G, A95C, H99C, S100C, L104I, T106S, and A108C, relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a S93G substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a glycine at position 93, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a glycine at position 93 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a V94I substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an isoleucine at position 94, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an isoleucine at position 94 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an H99C substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 99, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 99 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an S100A substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an alanine at position 100, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an alanine at position 100 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an S100C substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 100, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 100 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an L104I substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an isoleucine at position 104, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an isoleucine at position 104 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a T106G substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a glycine at position 106, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a glycine at position 106 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises an A108C substitution relative to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 108, where the positions are numbered according to SEQ ID NO: 279. In some embodiments, the DENV4 M protein comprises a cysteine at position 108 relative to a reference DENV4 M protein, where the positions are numbered by alignment of the amino acid sequence of the reference DENV4 M protein to SEQ ID NO: 279. ZIKV E Protein In some embodiments, the ZIKV E protein may comprise at least one mutation. In some embodiments, the ZIKV E protein comprises at least one substitution at one or more positions selected from D67, A69, T76, K93, L107, Q131, E133, T194, D197, S199, D200, H214, A248, T267, A270, A272, L273, S286, A319, H323, T325, R402, S403, K443, Q447, G450, A451, K454, S503, or A504, relative to the ZIKV E protein reference sequence (SEQ ID NO: 282). In some embodiments, the ZIKV E protein comprises at least one substitution selected from D67N, A69S, T76K, T76Q, T76Y, L107D, L107R, L107Q, Q131N, S199K, T267L, A270G, A272C, L273I, S286C, S403E, K454R, V494G, L495N, I496P, F497D, L498H, S499R, T500E, A501M, A501G, A501P, V502G, S503G, S503E, S503P, A504G, A504T, A504P, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at D67N and A69S relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, V494G, L495N, I496P, F497D, L498H, S499R, T500E, A501M, V502G, S503E, and A504T, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107D, S286C, V494G, L495N, I496P, F497D, L498H, S499R, T500E, A501M, V502G, and S503E, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at L107R, S403E, and K454R, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at L107R, S286C, S403E, and K454R, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Q, L107R, T267L, A270G, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Q, L107R, T267L, A270G, A272C, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Q, L107R, T267L, A270G, A272C, S286C, A501P, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Q, L107R, Q131N, T267L, A270G, A272C, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107R, Q131N, A270G, A272C, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, A272C, L273I, S286C, A501G, V502G, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, L273I, S286C, V502G, S503P, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, L273I, S286C, V502G, S503G, and A504P, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, L107Q, Q131N, A272C, L273I, S286C, V502G, S503G, and A504P, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76Y, L107Q, Q131N, S199K, A272C, L273I, S286C, V502G, S503G, and A504P, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, G106S, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, G106T, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at , relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at T76K, G106N, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at D67N, A69S, T76K, G106S, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at D67N, A69S, T76K, G106T, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises substitutions at D67N, A69S, T76K, G106N, L107D, S286C, S503G, and A504G, relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a D67N substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 67, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 67 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A69S substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a serine at position 69, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a serine at position 69 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T76K substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 76, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 76 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T76Q substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 76, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 76 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T76Y substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a tyrosine at position 76, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a tyrosine at position 76 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L107D substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an aspartic acid at position 107, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an aspartic acid at position 107 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L107R substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 107, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 107 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L107Q substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 107, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamine at position 107 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a Q131N substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 131, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 131 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S199K substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 199, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a lysine at position 199 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T267L substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a leucine at position 267, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a leucine at position 267 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A270G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 270, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 270 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A272C substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a cysteine at position 272, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a cysteine at position 272 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L273I substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an isoleucine at position 273, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an isoleucine at position 273 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S286C substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a cysteine at position 286, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a cysteine at position 286 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S403E substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 403, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 403 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a K454R substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 454, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 454 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a V494G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 494, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 494 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L495N substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 495, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an asparagine at position 495 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an I496P substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 496, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 496 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an F497D substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an aspartic acid at position 497, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an aspartic acid at position 497 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an L498H substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a histidine at position 498, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a histidine at position 498 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S499R substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 499, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an arginine at position 499 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a T500E substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 500, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 500 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A501M substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a methionine at position 501, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a methionine at position 501 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A501G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 501, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 501 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A501P substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 501, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 501 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a V502G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 502, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 502 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S503G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 503, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 503 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S503E substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 503, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glutamic acid at position 503 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an S503P substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 503, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 503 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A504G substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 504, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a glycine at position 504 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A504T substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a threonine at position 504, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a threonine at position 504 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises an A504P substitution relative to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 504, where the positions are numbered according to SEQ ID NO: 282. In some embodiments, the ZIKV E protein comprises a proline at position 504 relative to a reference ZIKV E protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV E protein to SEQ ID NO: 282. ZIKV M Protein In some embodiments, the ZIKV M protein may comprise at least one mutation. In some embodiments, the ZIKV M protein comprises at least one substitution at one or more positions selected from A95, V96, T97, L98, P99, H101, S102, R104, L106, Q107, T108, R109, S110, Q111, E115, S116, Y119, K121, I124, E127, N128, R132, A167, Y168, or S169, relative to the ZIKV M protein reference sequence (SEQ ID NO: 281). In some embodiments, the ZIKV M protein comprises at least one substitution selected from A95G, A95S, V96I, V96L, T97F, H101C, S102A, T108S, S110C, E115S, Y119H, A167C, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, S102A, S110C, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95S, V96I, S110C, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at S102A, S110C, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, S110C, E115S, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, S110C, E115S, Y119H, and A167C, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, H101C, S110C, E115S, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, H101C, S110C, E115S, Y119H, and A167C, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at V96L, T97F, H101C, S102A, T108S, S110C, E115S, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, S102A, T108S, S110C, E115S, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, H101C, S102A, T108S, S110C, E115S, and Y119H, relative to SEQ ID NO: 281.. In some embodiments, the ZIKV M protein comprises substitutions at A95G, V96I, S102A, S110C, E118D, and Y119H, relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an A95G substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a glycine at position 95, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a glycine at position 95 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an A95S substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 95, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 95 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a V96I substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an isoleucine at position 96, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an isoleucine at position 96 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a V96L substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a leucine at position 96, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a leucine at position 96 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a T97F substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a phenylalanine at position 97, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a phenylalanine at position 97 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an H101C substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 101, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 101 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an S102A substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an alanine at position 102, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an alanine at position 102 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a T108S substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 108, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 108 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an S110C substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 110, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 110 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an E115S substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 115, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a serine at position 115 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a Y119H substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a histidine at position 119, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a histidine at position 119 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises an A167C substitution relative to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 167, where the positions are numbered according to SEQ ID NO: 281. In some embodiments, the ZIKV M protein comprises a cysteine at position 167 relative to a reference ZIKV M protein, where the positions are numbered by alignment of the amino acid sequence of the reference ZIKV M protein to SEQ ID NO: 281. Flavivirus Proteins – Additional Amino Acid Substitution Positions In some embodiments, the flavivirus E protein may comprise at least one mutation. In some embodiments, the flavivirus E protein comprises at least one substitution at one or more positions selected from (i) T76, R93, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to SEQ ID NOs: 274, 276 or 280; or (ii) T76, K93, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A493, relative to SEQ ID NO: 278; or (iii) T76, K93, L107, Q131, E133, T194, D197, S199, D200, H214, A248, T267, A270, A272, L273, S286, A319, H323, T325, R402, S403, K443, Q447, G450, A451, K454, S503, or A504, relative to SEQ ID NO: 282. In some embodiments, the flavivirus M protein may comprise at least one mutation. In some embodiments, the flavivirus M protein comprises at least one substitution at one or more positions selected from (i) S93, V94, A95, L96, A97, H99, V100, L102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or A167, relative to SEQ ID NO: 273; or (ii) S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to SEQ ID NO: 275 or 277; or (iii) S93, V94, A95, L96, T97, H99, S100, M102, L104, E105, T106, R107, A108, E109, S113, S114, A117, K119, Q122, E125, S126, R130, S165, Y166, or G167, relative to SEQ ID NO: 279; or (iv) A95, V96, T97, L98, P99, H101, S102, R104, L106, Q107, T108, R109, S110, Q111, E115, S116, Y119, K121, I124, E127, N128, R132, A167, Y168, or S169, relative to SEQ ID NO: 281. Percent Identity In some embodiments, the compositions of the present disclosure include mRNA that encodes a flavivirus protein comprising an amino acid substitution at positions 107 and 76 corresponding to DENV2 E protein (e.g., SEQ ID NO: 276), and may comprise at least one additional amino acid substitution. Such proteins may possess one or more substitutions, deletions, and/or insertions at certain positions within its amino acid sequence, as compared to a naturally occurring or reference amino acid sequence. In some embodiments, a protein variant has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to a naturally occurring or reference sequence, and maintain the stabilized heterodimer formation that induces optimal immune responses. Global sequence alignment and local sequence alignment are two common methods used to compare and analyze sequences of DNA, RNA, or protein. Global sequence alignment compares the entire length of two sequences and finds the best possible alignment of the entire length of the sequences. It is useful, for example, when the two sequences being compared are similar in length and share significant homology. Local sequence alignment, on the other hand, identifies regions of similarity between sequences, allowing for gaps and mismatches in the alignment. This method is useful for identifying short regions of homology within larger sequences, and can be used to identify functional domains, protein families, and binding sites. Local alignment can be computationally more efficient than global alignment, and can be applied to sequences of different lengths. Unless stated otherwise herein, “percent (%) identity” between two mRNA polynucleotides or between two proteins refers to percent (%) identity determined using a global sequence alignment, comparing the length of two comparable sequences (e.g., E protein to a second E protein, M protein to a second M protein, mRNA encoding an E protein to mRNA encoding a second E protein (ORF, or entire mRNA), or mRNA encoding an M protein to mRNA encoding a second M protein (ORF or entire mRNA). Methods and computer programs for the alignment are well known in the art. It is understood that 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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular naturally occurring or reference sequence as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include but are not limited to those of the BLAST suite (Altschul, S.F., et al. Nucleic Acids Res.1997;25:3389-3402); and those based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. J. Mol. Biol.1981;147:195- 197). A general global alignment technique based on dynamic programming is the Needleman– Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. J. Mol. Biol.1920;48:443-453). A Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) also has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman–Wunsch algorithm. Other non- limiting examples of global alignment tools include Needle (from EMBOSS), Clustal Omega, MUSCLE (Multiple Sequence Comparison by Log-Expectation), MAFFT (Multiple Alignment using Fast Fourier Transform), and T-Coffee (Tree-based Consistency Objective Function for Alignment Evaluation). In some embodiments, an mRNA comprises an open reading frame that encodes a DENV protein comprising the amino acid sequence of any one of the sequences provided herein or comprising an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence of any one of the sequences provided herein. See, e.g., SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302- 346, and 353-363. In some embodiments, an mRNA comprises an open reading frame that encodes a ZIKV protein comprising the amino acid sequence of any one of the sequences provided herein or comprising an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence of any one of the sequences provided herein. See, e.g., SEQ ID NOs: 22- 25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352. In some embodiments, a vaccine of the present disclosure comprises an mRNA comprising an open reading frame encoding a YFV protein, wherein the YFV protein comprises the amino acid sequence of any one of the sequences provided herein or comprises an amino acid substitution at position 107 and 76, corresponding to the DENV2 E protein amino acid sequence, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238- 245, 187-188, and 214. Polynucleotides and polypeptides containing substitutions, insertions and/or deletions (e.g., indels), and covalent modifications with respect to naturally occurring or reference sequence, for example, the polypeptide (e.g., protein) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysine(s), can be added to polypeptide sequences (e.g., at the N-terminal and/or C-terminal end). Sequence tags can be used for peptide detection, purification and/or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the N-terminal and/or C-terminal regions of the amino acid sequence of a protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal amino acids) may be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence that is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., foldon regions) and the like are substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to further improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks are replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites are removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an mRNA vaccine. It will be understood that protein variants of stabilized heterotetramers comprising substitutions at amino acid positions 107 and 76 corresponding to a DENV2 E reference sequence will maintain the stabilized heterotetramer confirmation such that an appropriate immune response can be generated. Non-limiting examples of protein variants are provided in Tables 1-4. Signal Peptides In some embodiments, an mRNA has an open reading frame that encodes a signal peptide fused to the flavivirus protein. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane. A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15- 20 amino acids. Signal peptides from heterologous genes (which regulate expression of genes other than flavivirus proteins in nature) are known in the art and can be tested for desired properties and then incorporated into a protein or nucleic acid of the disclosure. In some embodiments, a signal peptide used in the present disclosure comprises MLRLLLRHHFHCLLLCAVWATPCLA (SEQ ID NO: 271). In some embodiments, a signal peptide used in the present disclosure comprises MKAILVVLLYTFTTANA (SEQ ID NO: 272). Fusion Proteins Some aspects of the present disclosure provide fusion proteins. In some embodiments, an mRNA encodes a fusion protein (e.g., a fusion protein comprising a flavivirus E protein and a flavivirus M protein). Thus, an encoded protein may include two or more proteins (e.g., protein and/or protein fragment) joined together with or without a linker. Fusion proteins, in some embodiments, retain the functional property of each independent (nonfusion) protein. In some embodiments, a fusion protein comprises 2, 3, 4, 5, 6, or 7 of the following flavivirus proteins: prM protein, M protein, C protein, and E protein. In some embodiments, a fusion protein comprises 2, 3, 4, 5, 6, or 7 of the following flavivirus proteins: prM protein, M protein, E protein, and C protein. In some embodiments, a fusion protein comprises 2, 3, 4, 5, 6, or 7 of the following flavivirus proteins: prM protein, M protein, E protein, C protein, NS1 protein, NS2A protein, NS2B protein, NS4A protein, NS4B protein, NS3 protein, and NS5 protein. Linkers and Cleavable Peptides In some embodiments, an mRNA that encodes a flavivirus protein of the disclosure further encodes a linker located between at least one or each protein of the fusion protein. In some embodiments, the linker is a GS linker. GS linkers are polypeptide linkers that include glycine and serine amino acids repeats. They comprise flexible and hydrophilic residues and can be used to perform fusion of protein subunits without interfering in the folding and function of the protein domains, and without formation of secondary structures. In some embodiments, an mRNA encodes a fusion protein that comprises a GS linker that is 3 to 20 amino acids long. For example, the GS linker may have a length of (or have a length of at least) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, a GS linker is (or is at least) 15 amino acids long (e.g., GGSGGSGGSGGSGGG (SEQ ID NO: 155)). In some embodiments, a GS linker is (or is at least) 8 amino acids long (e.g., GGGSGGGS (SEQ ID NO: 149)). In some embodiments, a GS linker is (or is at least) 7 amino acids long (e.g., GGGSGGG (SEQ ID NO: 150)). In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GGGS (SEQ ID NO: 151)). In some embodiments, the GS linker comprises (GGGS)n (SEQ ID NO: 152), where n is any integer from 1-5. In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GSGG (SEQ ID NO: 153)). In some embodiments, the GS linker comprises (GSGG)n (SEQ ID NO: 154), where n is any integer from 1-5. In some embodiments, a linker is a glycine linker, for example having a length of (or a length of at least) 3 amino acids (e.g., GGG). In some embodiments, the linker comprises GGGG (SEQ ID NO: 364). In some embodiments, the linker comprises GGPG (SEQ ID NO: 365). In some embodiments, a protein encoded by an mRNA includes two or more linkers, which may be the same or different from each other. The linker may be, for example, a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof (see, e.g., WO 2017/127750). This family of self- cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see, e.g., Kim, J.H. et al. PLoS ONE 2011;6:e18556). In some embodiments, the linker is an F2A linker. The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). Stabilization Domains Protein stabilization domains are protein sequences or structures that can enhance the stability of a protein to various environmental stresses, such as temperature, pH, and proteolysis. Non-limiting examples of protein stabilization domains for use to stabilize a flavivirus protein expressed by an mRNA include: lumazine synthase, ferritin, and thioredoxin. In some embodiments, a flavivirus protein is fused to a lumazine synthase. Lumazine synthase is protein from bacteria and plants that can stabilize fusion partners by forming homodimers or oligomers, which can enhance the solubility and stability of the target protein. In some embodiments, a flavivirusprotein is fused to ferritin. Ferritin is a protein found in animals, plants, and bacteria that can form a cage-like structure that can store and sequester iron ions, protecting the cell from oxidative damage. Fusion of a target protein with ferritin can improve its stability and solubility. In some embodiments, a flavivirus protein, e.g., an E protein, is fused to thioredoxin. Thioredoxin is small protein found in bacteria and eukaryotes that can act as a reducing agent and stabilize proteins by forming disulfide bonds. Nucleic Acids Encoding Flavivirus Proteins In some embodiments, any one of the engineered flavivirus proteins or flavivirus fusion proteins may be encoded by nucleic acids. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribo configuration, α-LNA having an α- L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- α-LNA having a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimeras and/or combinations thereof. mRNA of the present disclosure comprises an open reading frame (ORF) encoding at least one flavivirus protein. In some embodiments, the mRNA further comprises a 5 ^ untranslated region (UTR), 3 ^ UTR, a poly(A) tail and/or a 5 ^ cap analog. Preferred mRNA molecules are chemically modified. Messenger RNA Messenger RNA (mRNA) is RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. It is understood that mRNA is not self-amplifying RNA (saRNA) (see, e.g., Bloom K et al. Gene Therapy 2021; 28: 117–129 for a comparison of mRNA and saRNA). saRNAs include alphavirus replicase sequences that encode an RNA-dependent RNA polymerase. mRNA does not include alphavirus replicase sequences. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.” Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to, UTRs at their 5′-end (5′ UTR) and/or at their 3′-end (3′ UTR), in addition to other structural features, such as a 5′-cap structure or a 3′-poly(A) tail. Both the 5′ UTR and the 3′ UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5′-cap and the 3′-poly(A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. Untranslated Regions (UTRs) The mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. A “5′ untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. A “3′ untranslated region” (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. When RNA transcripts are being generated, the 5’ UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure. Where mRNAs are designed to encode a (at least one) viral protein, the mRNA may comprise a 5’ UTR and/or 3’ UTR. UTRs of an mRNA are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is a growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5’ UTR and 3’ UTR sequences are known. It should also be understood that the mRNA of the present disclosure may include any 5’ UTR and/or any 3’ UTR. Exemplary UTR sequences include SEQ ID NOs: 256-270; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein. In some embodiments, a 5' UTR of the present disclosure comprises a sequence selected from: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 256), GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC (SEQ ID NO: 257), GAGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAA CUAGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 258), and GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAACU AGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 259). In some embodiments, a 3' UTR of the present disclosure comprises a sequence selected from UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 260), UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 261), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGACUAGUGGUCUUUGA AUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 262), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGGAUUGAGACUACGGGUGGUCUUUGAAUAAAG UCUGAGUGGGCGGC (SEQ ID NO: 263), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGCAUAGACACUACGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC (SEQ ID NO: 264), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC (SEQ ID NO: 265), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGACGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGC (SEQ ID NO: 266), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 267), and UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 268). In some embodiments, a 3′ UTR comprises, in 5′-to-3′ order: (a) the nucleic acid sequence UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAG (SEQ ID NO: 269), (b) an identification and ratio determination (IDR) sequence, and (c) the nucleic acid sequence UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 270). IDR sequences are described herein in the section entitled “Identification and Ratio Determination (IDR) Sequences.” UTRs may also be omitted from the mRNA provided herein. A 5 ^ UTR does not encode a protein (is non-coding). Natural 5′ UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5′UTR also have been known to form secondary structures which are involved in elongation factor binding. In some embodiments of the disclosure, a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF. In other embodiments, a 5’ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5’ UTRs include Xenopus or human derived a-globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, US9012219). CMV immediate-early 1 (IE1) gene (US2014/0206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 232) (WO2014/144196) may also be used. In other embodiments, a 5' UTR is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO2015/101414, WO2015/101415, WO2015/062738, WO2015/024667, WO2015/024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO/2015101414, WO2015101415, WO/2015/062738), 5' UTR element derived from the 5' UTR of an hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015/024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR. A 3 ^ UTR does not encode a protein (is non-coding). Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3′ UTR AU rich elements (AREs) can be used to modulate the stability of mRNA of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 1 day, 2 days, and 7 days post-transfection. Those of ordinary skill in the art will understand that 5’ UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence. For example, a heterologous or synthetic 5’ UTR may be used with a synthetic 3’ UTR or with a heterologous 3’ UTR. Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels. Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US2010/0293625 and WO2015/085318A2, each of which is herein incorporated by reference. It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ UTR or 5′ UTR may be altered relative to a wild-type/native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR. In some embodiments, a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3′ UTR may be used as described in US2010/0129877, which is incorporated herein by reference. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern. The untranslated region may also include translation enhancer elements (TEE). As a non- limiting example, the TEE may include those described in US 2009/0226470, herein incorporated by reference, and those known in the art. Open Reading Frames An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5′ and/or 3′ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an mRNA of the present disclosure. 5’ End Capping In some embodiments, an mRNA comprises a 5′ terminal cap.5′-capping of polynucleotides may be completed concomitantly during an in vitro transcription reaction using, for example, 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 mRNA may be completed post-transcriptionally using, for example, 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′-O methyl-transferase 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′-O 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′-O methyl-transferase. Enzymes may be derived from a recombinant source. Other cap analogs may be used. Polyadenylation Tailing A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. It can, in some instances, comprise up to about 400 adenine nucleotides. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation. In some embodiments, the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA. In some embodiments, a poly(A) tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides. In some embodiments, a poly(A) tail has a length of 100 nucleotides. Additional Stabilizing Elements mRNA provided herein, in some embodiments, includes an additional stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3'-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5’ and two nucleotides 3′ relative to the stem-loop. In some embodiments, an mRNA includes an open reading frame (coding region), a histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g., Luciferase, GFP, EGFP, β-Galactosidase, EGFP), or a marker or selection protein (e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)). In some embodiments, an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, they act synergistically to increase the protein expression beyond the level observed with either of the individual elements. The synergistic effect of the combination of poly(A) and a histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence. In some embodiments, an mRNA does not include a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3′ of naturally-occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not include an intron. An mRNA may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides. In some embodiments, an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3 ’UTR. The AURES may be removed from the mRNA. Alternatively, the AURES may remain in the mRNA. Sequence Optimization In some embodiments, an open reading frame encoding a protein of the disclosure is codon optimized. Codon optimization methods are known in the art. An open reading frame of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art – non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame sequence is optimized using optimization algorithms. Chemically Unmodified Nucleotides In some embodiments, an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT). Chemically Modified Nucleotides The compositions of the present disclosure comprise, in some embodiments, a nucleic acid molecule encoding a flavivirus stabilized heterotetramer protein, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally-occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art. In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally-occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database. In some embodiments, a non-naturally-occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally-occurring modified nucleotides and nucleosides can be found, inter alia, in international publication numbers WO2013052523A1; WO2014093924A1; WO2015051173A2; WO2015051169A2; WO2015089511A2; or WO2017153936A1, each of which is herein incorporated by reference in its entirety. Hence, nucleic acids of the disclosure (e.g., DNA and RNA, such as mRNA) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof. Nucleic acids of the disclosure (e.g., DNA and RNA, such as mRNA), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides. In some embodiments, a modified mRNA introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. In some embodiments, a modified mRNA introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. Nucleic acids (e.g., RNA, such as mRNA), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide 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 nucleic acid may be chemically modified. The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA, such as mRNA). 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 non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides. 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 bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. 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 nucleic acids of the present disclosure. In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy- uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (ψ). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5- methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications. In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise N1-methyl-pseudouridine (m1ψ), N1-ethyl-pseudouridine (e1ψ), 5-methoxy- uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and/or pseudouridine (ψ). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5- methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the RNA includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications. In some embodiments, a mRNA comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the mRNA. In some embodiments, the ORF comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of m1ψ, adenosine, guanosine, and cytidine. In some embodiments, the ORF comprises nucleosides consisting of m1ψ, adenosine, guanosine, and cytidine. In some embodiments, a mRNA comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the mRNA. In some embodiments, the ORF comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of m1ψ, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, the ORF comprises nucleosides consisting of m1ψ, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, a mRNA comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the mRNA. In some embodiments, the ORF comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of ψ, adenosine, guanosine, and cytidine. In some embodiments, the ORF comprises nucleosides consisting of ψ, adenosine, guanosine, and cytidine. In some embodiments, a mRNA comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all cytidine positions of the mRNA. In some embodiments, the ORF comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of ψ, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, the ORF comprises nucleosides consisting of ψ, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, a mRNA comprises uridine at one or more or all uridine positions of the mRNA. In some embodiments, the ORF comprises uridine at one or more or all uridine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of uridine, adenosine, guanosine, and cytidine. In some embodiments, the ORF comprises nucleosides consisting of uridine, adenosine, guanosine, and cytidine. In some embodiments, a mRNA comprises 5-methyl-uridine and 5-methyl cytidine at one or more or all uridine and cytidine positions, respectively, of the mRNA. In some embodiments, the ORF comprises 5-methyl-uridine substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of 5-methyl- uridine, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, the ORF comprises nucleosides consisting of 5-methyl-uridine, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, a mRNA comprises 5-methyl-uridine and 5-methyl cytidine at one or more or all uridine and cytidine positions, respectively, of the mRNA. In preferred embodiments, RNAs (e.g., mRNAs) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid 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. The nucleic acids 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 nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly(A) tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C. The nucleic acid 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% to 80%, 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. The mRNA 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 nucleic acids 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 nucleic acid 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 nucleic acid 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). Identification and Ratio Determination (IDR) Sequences An Identification and Ratio Determination (IDR) sequence is a sequence of a biological molecule (e.g., nucleic acid or protein) that, when combined with the sequence of a target biological molecule, serves to identify the target biological molecule. Typically, an IDR sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and can be used as a reference to identify the target molecule. Thus, in some embodiments, a nucleic acid (e.g., mRNA) comprises (i) a target sequence of interest (e.g., a coding sequence encoding an antigenic peptide or protein); and (ii) a unique IDR sequence. An RNA species (e.g., RNA having a given coding sequence) may comprise an IDR sequence that differs from the IDR sequence of other RNA species (e.g., RNA(s) having different coding sequence(s)). Each IDR sequence thus identifies a particular RNA species, and so the abundance of IDR sequences may be measured to determine the abundance of each RNA species in a composition. Use of distinct IDR sequences to identify RNA species allows for analysis of multivalent RNA compositions (e.g., containing multiple RNA species) containing RNA species with similar coding sequences and/or lengths, which could otherwise be difficult to distinguish using PCR- or chromatography-based analysis of full-length RNAs. Each RNA species in a multivalent RNA composition may comprise an IDR sequence that is not a sequence isomer of an IDR sequence of another RNA species in a multivalent RNA composition (e.g., the IDR sequence does not have the same number of adenosine nucleotides, the same number of cytosine nucleotides, the same number of guanine nucleotides, and the same number of uracil nucleotides, as another IDR sequence in the composition, even if those sequences have different sequences). Having identical nucleotide compositions causes sequence isomers to have the same mass, presenting a challenge to distinguishing sequence isomers using mass-based identification methods (e.g., mass spectrometry). Each RNA species in a multivalent RNA composition may comprise an IDR sequence having a mass that differs from the mass of IDR sequences of each other RNA species in a multivalent RNA composition. For example, the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da. Use of IDR sequences with distinct masses allows RNA fragments comprising different IDR sequences to be distinguished using mass-based analysis methods (e.g., mass spectrometry), which do not require reverse transcription, amplification, or sequencing of RNAs. Each RNA species in an RNA composition may comprises an IDR sequence with a different length. For example, each IDR sequence may have a length independently selected from 0 to 25 nucleotides. The length of a nucleic acid influences the rate at which the nucleic acid traverses a chromatography column, and so the use of IDR sequences of different lengths on different RNA species allows RNA fragments having different IDR sequences to be distinguished using chromatography-based methods (e.g., LC-UV). IDR sequences may be chosen such that no IDR sequence comprises a start codon, ‘AUG’. Lack of a start codon in an IDR sequence prevents undesired translation of nucleotide sequences within and/or downstream from the IDR sequence. IDR sequences may be chosen such that no IDR sequence comprises a recognition site for a restriction enzyme. In one example, no IDR sequence comprises a recognition site for XbaI, ‘UCUAG’. Lack of a recognition site for a restriction enzyme (e.g., XbaI recognition site ‘UCUAG’) allows the restriction enzyme to be used in generating and modifying a DNA template for in vitro transcription, without affecting the IDR sequence or sequence of the transcribed RNA or DNA plasmid. Nucleic Acid Production Chemical Synthesis Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences. The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase. In Vitro Transcription cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of mRNA is known in the art and is described in International Publication WO 2014/152027, which is incorporated by reference herein in its entirety. In some embodiments, the RNA of the present disclosure is prepared in accordance with any one or more of the methods described in WO 2018/053209 or WO 2019/036682, each of which is incorporated by reference herein. In some embodiments, the mRNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA, for example, but not limited to DENV mRNA. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest. Thus, in some embodiments, a DNA plasmid encoding an E protein and an M protein capable of forming the stabilized flavivirus heterotetramer described herein is provided. As used herein, “plasmid DNA” or “pDNA” refers to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently. In some embodiments, plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation). In some embodiments, plasmid DNA comprises an origin of replication, which may contain one or more heterologous nucleic acids, for example nucleic acids encoding therapeutic proteins that may serve as a template for RNA polymerase. Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by a restriction enzyme digest). In some embodiments, an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a poly(A) tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template. In some embodiments, a nucleic acid (e.g., template DNA and/or RNA) includes 200 to 3,000 nucleotides. For example, a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides. An in vitro transcription system typically comprises a transcription buffer (e.g., with magnesium), nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase (e.g., T7 RNA polymerase). In some embodiments, one or more of the NTPs is a chemically modified NTP (e.g., with 1-methylpseudouridine or other chemical modifications described herein and/or known in the art). In some embodiments, the NTPs comprise adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), or an analog of each respective NTP. The ratio of NTPs may vary. In some embodiments, the ratio of GTP:ATP:CTP:UTP is 1:1:1:1. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof. In some embodiments, the amount of the GTP is greater than the amount of the UTP. In some embodiments, the amount of ATP is greater than the amount of UTP, and the amount of CTP is greater than the amount of UTP. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof. In some embodiments, an IVT system comprises an at least 2:1 ratio of GTP concentration to ATP concentration, an at least 2:1 ratio of GTP concentration to CTP concentration, and an at least 4:1 ratio of GTP concentration to UTP concentration. In some embodiments, an IVT system comprises a 2:1 ratio of GTP concentration to ATP concentration, a 2:1 ratio of GTP concentration to CTP concentration, and a 4:1 ratio of GTP concentration to UTP concentration. In some embodiments, an IVT system comprises guanosine diphosphate (GDP). In some embodiments, an IVT system comprises an at least 3:1 ratio of GTP plus GDP concentration to ATP concentration, an at least 6:1 ratio of GTP plus GDP concentration to CTP concentration, and an at least 6:1 ratio of GTP plus GDP concentration to UTP concentration. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase. An IVT system, in some embodiments, comprises magnesium buffer, dithiothreitol (DTT) spermidine, pyrophosphatase, and/or RNase inhibitor. In some embodiments, an IVT system omits an RNase inhibitor. An IVT system may be incubated at 25 degrees Celsius or at 37 degrees Celsius. Other temperatures may be used, depending in part on the polymerase (e.g., use of a variant polymerase). In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp. Purification Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. Protein Synthesis Any one of the proteins described herein may be synthesized using methods known in the art. For example, the proteins may be synthesized in vitro using a cell-free protein synthesis system (e.g., in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis). Following synthesis, in some embodiments, the proteins are purified (e.g., using affinity, ion exchange, hydrophobic interaction, and/or size exclusion) and then administered using techniques known in the art. Lipid Compositions In some embodiments, the nucleic acids are in (e.g., formulated as) a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and/or a lipoplex. In preferred embodiments, nucleic acids of the present disclosure are in (e.g., formulated as) lipid nanoparticle (LNP) compositions. Lipid nanoparticles typically comprise amino lipid, non- cationic lipid, structural lipid, and PEG lipid components along with the nucleic acid cargo (e.g., RNA, such as mRNA) of interest. A lipid nanoparticles of the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575; PCT/US2016/069491; PCT/US2016/069493; and PCT/US2014/066242, all of which are incorporated by reference herein in their entirety. In some embodiments, a lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)- modified lipid. In some embodiments, a lipid nanoparticle comprises 20-60 mole percent (mol%) ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid. In some embodiments, a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-30 mol% non-cationic lipid, 10-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid. In some embodiments, a lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%. In some embodiments, a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid. For example, a lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 45-55 mol% ionizable amino lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid. Ionizable amino lipids In some embodiments, the ionizable lipid is a compound of Formula (IL*)
Figure imgf000153_0001
(IL*) or a salt thereof, wherein: R1 is -OH, -NRN-C4-10 cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN’’); RN is H or C1-6 alkyl; RN’ is H or C1-6 alkyl; RN’’ is H or C1-6 alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8; M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2; M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3; R2 is
Figure imgf000153_0002
or –(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl; R2a is -H or C1-10 alkyl; R2b is -H or C1-10 alkyl; R2c is C1-8 alkyl or C2-8 alkenyl; R3 is ; R3a is H or C1-10 alkyl; R3b is H or C1-8 alkyl; and R3c is C1-10 alkyl or C2-8 alkenyl. In some embodiments, the ionizable lipid is of Formula (IL**-I):
Figure imgf000154_0001
(IL**-I) or a salt thereof, wherein: R1 is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2c is C4-8 alkyl; R3a is C7-10 alkyl; and R3c is C3-5 alkyl. In some embodiments, the ionizable lipid is of Formula (IL**-III):
Figure imgf000154_0002
(IL**-III) or a salt thereof, wherein: R1 is NRN-C4-10 cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN’’); RN is H; RN’ is C1-2 alkyl; RN’’ is H; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2a is C7-10 alkyl; R2c is C4-6 alkyl; R3a is C1-3 alkyl; and R3c is C4-6 alkyl. In some embodiments, the ionizable lipid is of Formula (IL**-IV):
Figure imgf000155_0001
(IL**-IV) or a salt thereof, wherein: R1 is OH; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2b is C3-5 alkyl; R2c is C2-4 alkyl; R3a is C7-10 alkyl; and R3c is C4-6 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-I): (IL*-Ia) or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*; and R3a is C1-8 alkyl. In some embodiments, ionizable lipid is of Formula (IL*-Ia):
Figure imgf000156_0001
or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for Formula IL*; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-Ia’):
Figure imgf000156_0002
or a salt thereof, wherein: o, M, M’, R2c and R3c are as defined for variable IL*; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIa):
Figure imgf000156_0003
(IL*-IIa) or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for Formula IL*; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-II’):
Figure imgf000157_0001
(IL*-II’) or a salt thereof, wherein: o, M, M’, R2c and R3c are as defined for variable IL*; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-III):
Figure imgf000157_0002
(IL*-III) or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*; R2a is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):
Figure imgf000157_0003
or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*; R2b is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):
Figure imgf000158_0001
or a salt thereof, wherein: R1, o, M, M’, R2c, and R3c are as defined for variable IL*; R2a is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’):
Figure imgf000158_0002
(IL*-IIIa’) or a salt thereof, wherein: R1, o, M, M’, R2c, and R3c are as defined for variable IL*; R2a is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIb):
Figure imgf000158_0003
(IL*-IIIb) or a salt thereof, wherein: R1, o, M, M’, R2c, and R3c are as defined for variable IL*; R2a is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’):
Figure imgf000159_0001
(IL*-IIIb’) or a salt thereof, wherein: R1, o, M, M’, R2c, and R3c are as defined for variable IL*; R2a is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IV):
Figure imgf000159_0002
(IL*-IV) or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*; R2b is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IVa):
Figure imgf000159_0003
(IL*-IVa) or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*; R2b is a C1-8 alkyl; and R3a is C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-Iva’):
Figure imgf000160_0001
(IL*-IVa) or a salt thereof, wherein: o, M, M’, R2c, and R3c are as defined for variable IL*; R2a is a C1-8 alkyl; and R3a is C1-8 alkyl. Variables o, R1, RN, RN’, RN’’ of Ionizable Lipid In some embodiments of the ionizable lipid, o is 1. In some embodiments of the ionizable lipid, o is 2. In some embodiments of the ionizable lipid, o is 3. In some embodiments of the ionizable lipid, o is 4. In some embodiments of the ionizable lipid, R1 is -OH. In some embodiments of the ionizable lipid, RN is H. In some embodiments of the ionizable lipid, RN is methyl. In some embodiments of the ionizable lipid, RN is ethyl. In some embodiments of the ionizable lipid, R1 is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RN’RN’’). In some embodiments of the ionizable lipid, RN’ is H. In some embodiments of the ionizable lipid, RN’ is methyl. In some embodiments of the ionizable lipid, RN’ is ethyl. In some embodiments of the ionizable lipid, RN’’ is H. In some embodiments of the ionizable lipid, RN’’ is methyl. In some embodiments of the ionizable lipid, RN’’ is ethyl. In some embodiments of the ionizable lipid, RN’ is H and RN’’ is methyl. In some embodiments of the ionizable lipid, In some embodiments of the ionizable lipid,
Figure imgf000161_0001
Variables m and n of the Ionizable Lipid In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, m is 5. In some embodiments of the ionizable lipid, m is 6. In some embodiments of the ionizable lipid, m is 7. In some embodiments of the ionizable lipid, m is 8. In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, n is 5. In some embodiments of the ionizable lipid, n is 6. In some embodiments of the ionizable lipid, n is 7. In some embodiments of the ionizable lipid, n is 8. In some embodiments of the ionizable lipid, n is 5 and m is 7. In some embodiments of the ionizable lipid, n is 7 and m is 7. In some embodiments of the ionizable lipid, m is 6 and n is 6. Variables M and M’ In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2. In some embodiments of the ionizable lipid, M is -C(=O)-O-* wherein * indicates attachment to R2. In some embodiments of the ionizable lipid, M’ is -O-C(=O)-*, wherein * indicates attachment to R3. In some embodiments of the ionizable lipid, M’ is -C(=O)-O-* wherein * indicates attachment to R3. In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2, and M’ is -C(=O)-O-* wherein * indicates attachment to R3 Variables R2, R2a, R2b, R2c In some embodiments of the ionizable lipid, R2 is
Figure imgf000162_0001
. In some embodiments of the ionizable lipid, R2a is hydrogen. In some embodiments of the ionizable lipid, R2a is methyl. In some embodiments of the ionizable lipid, R2a is ethyl. In some embodiments of the ionizable lipid, R2a is propyl. In some embodiments of the ionizable lipid, R2a is butyl. In some embodiments of the ionizable lipid, R2a is pentyl. In some embodiments of the ionizable lipid, R2a is hexyl. In some embodiments of the ionizable lipid, R2a is heptyl. In some embodiments of the ionizable lipid, R2a is octyl. In some embodiments of the ionizable lipid, R2b is hydrogen. In some embodiments of the ionizable lipid, R2b is methyl. In some embodiments of the ionizable lipid, R2b is ethyl. In some embodiments of the ionizable lipid, R2b is propyl. In some embodiments of the ionizable lipid, R2b is butyl. In some embodiments of the ionizable lipid, R2b is pentyl. In some embodiments of the ionizable lipid, R2b is hexyl. In some embodiments of the ionizable lipid, R2b is heptyl. In some embodiments of the ionizable lipid, R2b is octyl. In some embodiments of the ionizable lipid, R2a is hydrogen and R2b is hydrogen. In some embodiments of the ionizable lipid, R2a is hexyl and R2b is hydrogen. In some embodiments of the ionizable lipid, R2a is octyl and R2b is hydrogen. In some embodiments of the ionizable lipid, R2a is hydrogen and R2b is butyl. In some embodiments of the ionizable lipid, R2c is methyl. In some embodiments of the ionizable lipid, R2c is ethyl. In some embodiments of the ionizable lipid, R2c is propyl. In some embodiments of the ionizable lipid, R2c is butyl. In some embodiments of the ionizable lipid, R2c is pentyl. In some embodiments of the ionizable lipid, R2c is hexyl. In some embodiments of the ionizable lipid, R2c is heptyl. In some embodiments of the ionizable lipid, R2c is octyl. In some embodiments of the ionizable lipid, R2 is –(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl. In some embodiments of the ionizable lipid, R2 is –(C1-6 alkylene)-(cyclohexyl)-C1-6 alkyl. In some embodiments of the ionizable lipid, R2 is –(C1-6 alkylene)-(cyclopentyl)-C1-6 alkyl. Variables R3, R3a, R3b, and R3c
Figure imgf000163_0001
In some embodiments of the ionizable lipid, R3 is . In some embodiments of the ionizable lipid, R3a is hydrogen. In some embodiments of the ionizable lipid, R3a is methyl. In some embodiments of the ionizable lipid, R3a is ethyl. In some embodiments of the ionizable lipid, R3a is propyl. In some embodiments of the ionizable lipid, R3a is butyl. In some embodiments of the ionizable lipid, R3a is pentyl. In some embodiments of the ionizable lipid, R3a is hexyl. In some embodiments of the ionizable lipid, R3a is heptyl. In some embodiments of the ionizable lipid, R3a is octyl. In some embodiments of the ionizable lipid, R3b is hydrogen. In some embodiments of the ionizable lipid, R3b is methyl. In some embodiments of the ionizable lipid, R3b is ethyl. In some embodiments of the ionizable lipid, R3b is propyl. In some embodiments of the ionizable lipid, R3b is butyl. In some embodiments of the ionizable lipid, R3b is pentyl. In some embodiments of the ionizable lipid, R3b is hexyl. In some embodiments of the ionizable lipid, R3b is heptyl. In some embodiments of the ionizable lipid, R3b is octyl. In some embodiments of the ionizable lipid, R3a is octyl and R3b is hydrogen. In some embodiments of the ionizable lipid, R3a is ethyl and R3b is hydrogen. In some embodiments of the ionizable lipid, R3a is hexyl and R3b is hydrogen. In some embodiments of the ionizable lipid, R3c is methyl. In some embodiments of the ionizable lipid, R3c is ethyl. In some embodiments of the ionizable lipid, R3c is propyl. In some embodiments of the ionizable lipid, R3c is butyl. In some embodiments of the ionizable lipid, R3c is pentyl. In some embodiments of the ionizable lipid, R3c is hexyl. In some embodiments of the ionizable lipid, R3c is heptyl. In some embodiments of the ionizable lipid, R3c is octyl. It is understood that, for an ionizable lipid, variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3c can each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o,.R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3c can be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3c. In some embodiments, the ionizable lipid is a compound selected from:
Figure imgf000164_0001
In some embodiments, the ionizable lipid is
Figure imgf000164_0002
In some embodiments, the ionizable lipid is
Figure imgf000164_0003
In some embodiments, the ionizable lipid is Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge. mRNA-Lipid Adduct Modules It has been determined that certain ionizable lipids are susceptible to the formation of lipid-polynucleotide adducts. In particular, ionizable lipids that comprise a tertiary amine group may decompose into one or both of a secondary amine and a reactive aldehyde species capable of interacting with polynucleotides (such as mRNA) to form an ionizable lipid-polynucleotide adduct impurity that can be detected by reverse phase ion pair chromatography (RP-IP HPLC). For example, oxidation of the tertiary amine may lead to N-oxide formation that can undergo acid/base-catalyzed hydrolysis at the amine to generate aldehydes and secondary amines which may form adducts with mRNA. Thus, in some aspects, the ionizable lipid-polynucleotide adduct impurity is an aldehyde-mRNA adduct impurity. It also has been determined that such adducts may disrupt mRNA translation and impact the activity of lipid nanoparticle (LNP) formulated mRNA products. Thus, it can be advantageous to prepare and use LNP compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity, such as wherein less than about 20%, less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid- polynucleotide adduct impurity, as may be measured by RP-IP HPLC. Thus, in accordance with some aspects, an LNP composition is provided wherein less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid-polynucleotide adduct impurity, including less than 10%, less than 5%, or less than 1%, as may be measured by RP-IP HPLC. In some aspects, an amount of lipid aldehydes in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of N- oxide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of transition metals, such as Fe, in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of alkyl halide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of anhydride compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of ketone compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of conjugated diene compounds in the composition is less than about 50 ppm, including less than 50 ppm. In some aspects, the composition is stable against the formation of ionizable lipid- polynucleotide adduct impurity. In some aspects, an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when stored at a temperature of about 25 °C or below, including at an average rate of less than 2% per day. In some aspects, an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a temperature of about 5 °C or below, including at an average rate of less than 0.5% per day. In some aspects, an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a refrigerated temperature, optionally wherein the refrigerated temperature is about 5 °C. Lipid vehicle (e.g., LNP) compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity can be prepared by methods that inhibit formation of one or both of N-oxides and aldehydes. Such methods may comprise treating a composition comprising an ionizable lipid comprising a tertiary amine group to inhibit formation of one or both of N-oxides and aldehydes, such as by treating the composition with a reducing agent; treating the composition with a chelating agent; adjusting the pH of the composition; adjusting the temperature of the composition; and adjusting the buffer in the composition. Such methods may comprise, prior to combining the ionizable lipid with a polynucleotide, one or more of treating the ionizable lipid with a scavenging agent; treating the ionizable lipid with a reductive treatment agent; treating the ionizable lipid with a reducing agent; treating the ionizable lipid with a chelating agent; treating the polynucleotide with a reducing agent; and treating the polynucleotide with a chelating agent. In accordance with any of the foregoing, the scavenging agent, reductive treatment agent, and/or reducing agent may be an agent that reacts with aldehyde, ketone, anhydride and/or diene compounds. A scavenging agent may comprise one or more selected from (O-(2,3,4,5,6- Pentafluorobenzyl)hydroxylamine hydrochloride) (PFBHA), methoxyamine (e.g., methoxyamine hydrochloride), benzyloxyamine (e.g., benzyloxyamine hydrochloride), ethoxyamine (e.g., ethoxyamine hydrochloride), 4-[2-(aminooxy)ethyl]morpholine dihydrochloride, butoxyamine (e.g., tert-butoxyamine hydrochloride), 4-Dimethylaminopyridine (DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), Triethylamine (TEA), Piperidine 4- carboxylate (BPPC), and combinations thereof. A reductive treatment agent may comprise a boron compound (e.g., sodium borohydride and/or bis(pinacolato)diboron). A reductive treatment agent may comprise a boron compound, such as one or both of sodium borohydride and bis(pinacolato)diboron). A chelating agent may comprise immobilized iminodiacetic acid. A reducing agent may comprise an immobilized reducing agent, such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof. A reducing agent may comprise a free reducing agent, such as potassium metabisulfite, sodium thioglycolate, tris(2-carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetyl cysteine, glutathione, dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, lipoic acid, or a combination thereof. In accordance with any of the foregoing, the pH may be, or adjusted to be, a pH of from about 7 to about 9 (for example, about 7, about 7.5, about 8, about 8.5, or about 9). In accordance with any of the foregoing, a buffer may be selected from sodium phosphate, sodium citrate, sodium succinate, histidine, histidine-HCl, sodium malate, sodium carbonate, and TRIS (tris(hydroxymethyl)aminomethane). In accordance with any of the foregoing, a buffer may be TRIS and may be, or adjusted to be, from about 20 mM to about 150 mM TRIS. In accordance with any of the foregoing, the temperature of the composition may be, or adjusted to be, 25 ⁰C or less. The composition may also comprise a free reducing agent or antioxidant. Non-cationic lipids In certain embodiments, a lipid nanoparticle described herein comprise one or more non- cationic lipids. Non-cationic lipids may be phospholipids. In some embodiments, a lipid nanoparticle comprises 5-25 mol% non-cationic lipid. For example, a lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, a lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid. In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof. In some embodiments, a lipid nanoparticle comprises 5 – 15 mol%, 5 – 10 mol%, or 10 – 15 mol% DSPC. For example, a lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC. In certain embodiments, the lipid composition of a lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., an mRNA) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some embodiments, a phospholipid of the present disclosure comprises 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof. In certain embodiments, a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially useful in the present disclosure is a compound of Formula (IX): or a salt thereof, wherein: each R1 is independently optionally substituted alkyl; or optionally two R1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula:
Figure imgf000170_0001
each instance of L2 is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), - NRNC(O)O, or NRNC(O)N(RN); each instance of R2 is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), - OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, - OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or - N(RN)S(O)2O; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2; provided that the compound is not of the formula: , wherein each instance of R2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl. In some embodiments, the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT/US2018/037922. In some embodiments, a lipid nanoparticle comprises 5-25 mol% non-cationic lipid relative to the other lipid components. For example, a lipid nanoparticle may comprise 5-30 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, or 25-30 mol% non-cationic lipid. In some embodiments, a lipid nanoparticle comprises a 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or 30 mol% non- cationic lipid. In some embodiments, a lipid nanoparticle comprises 5-25 mol% phospholipid relative to the other lipid components. For example, the lipid nanoparticle may comprise 5-30 mol%, 5- 15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, or 25-30 mol% phospholipid. In some embodiments, the lipid nanoparticle 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or 30 mol% phospholipid lipid. Structural Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term “structural lipid” includes sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in a lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol. In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No.16/493,814. In some embodiments, a lipid nanoparticle comprises 25-55 mol% structural lipid relative to the other lipid components. For example, a lipid nanoparticle may comprise 10- 55 mol%, 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid. In some embodiments, a lipid nanoparticle comprises 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid. In some embodiments, a lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 34-35 mol%, 35- 36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%. In some embodiments, a lipid nanoparticle comprises 25-55 mol% sterol. For example, a lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol. In some embodiments, a lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol. In some embodiments, a lipid nanoparticle comprises 35-40 mol% cholesterol. For example, a lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol. Polyethylene Glycol (PEG)-Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids. As used herein, the term “PEG-lipid” or “PEG-modified lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3- amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG- DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and/or PEG-DPG. In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG-lipid is PEG2k-DMG. In some embodiments, a lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE. PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety. In general, some of the other lipid components (e.g., PEG lipids) of various formulae described herein may be synthesized as described International Patent Application No. PCT/US2016/000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety. The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG- DMG has the following structure:
Figure imgf000173_0001
In some embodiments, PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy- PEGylated lipid comprises an –OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present disclosure. In certain embodiments, a PEG lipid useful in the present disclosure is a compound of Formula (X):
Figure imgf000174_0001
(X), or salts thereof, wherein: R3 is –ORO; RO is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L1 is optionally substituted C1-10 alkylene, wherein at least one methylene of the optionally substituted C1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN); D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula:
Figure imgf000174_0002
each instance of L2 is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), - NRNC(O)O, or NRNC(O)N(RN); each instance of R2 is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), - OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O) , OS(O), S(O)O, - OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or - N(RN)S(O)2O; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2. In certain embodiments, the compound of Fomula (X) is a PEG-OH lipid (i.e., R3 is – ORO, and RO is hydrogen). In certain embodiments, the compound of Formula (X) is of Formula (X-OH):
Figure imgf000175_0001
(X-OH), or a salt thereof. In certain embodiments, a PEG lipid useful in the present disclosure is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present disclosure is a compound of Formula (XI). Provided herein are compounds of Formula (XI):
Figure imgf000175_0002
or a salts thereof, wherein: R3 is–ORO; RO is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R5 is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), - C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), - C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, - OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, - N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (XI) is of Formula (XI-OH):
Figure imgf000176_0001
or a salt thereof. In some embodiments, r is 40-50. In yet other embodiments the compound of Formula (XI) is:
Figure imgf000176_0002
. or a salt thereof. In some embodiments, the compound of Formula (XI) is
Figure imgf000176_0003
In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid. In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. US15/674,872. In some embodiments, a lipid nanoparticle comprises 0.5-15 mol% PEG lipid relative to the other lipid components. For example, a lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% PEG lipid. In some embodiments, a lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG- lipid. In some embodiments, a lipid nanoparticle comprises 1-5% PEG-modified lipid, optionally 1-3 mol%, for example, 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%. In some embodiments, a lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid. For example, a lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, a lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid. Some embodiments comprise adding PEG to a composition comprising an LNP encapsulating a nucleic acid (e.g., which already includes PEG in the amounts listed above). Without being bound by theory, it is believed that spiking an LNP composition with additional PEG can provide benefits during lyophilization. Thus, some embodiments, comprise adding additional PEG as compared to an amount used for a non-lyophilized LNP composition. In embodiments comprise adding about 0.5mo% or more PEG to an LNP composition, such as about 1mol%, about 1.5mol%, about 2mol%, about 2.5mol%, about 3mol%, about 3.5mol%, about 4mol%, about 5mol%, or more after formation of an LNP composition (e.g., which already contains PEG in amount listed elsewhere herein). In some embodiments, a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid. In some embodiments, an LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG. In some embodiments, an LNP of the present disclosure comprises an ionizable amino lipid of any of Formula VI, VII or VIIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG. In some embodiments, an LNP of the present disclosure comprises an ionizable amino lipid of any of Formula VI, VII or VIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula XI. In some embodiments, an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula VIII, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI. In some embodiments, an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula IX, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI. In some embodiments, an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid having Formula IX, a structural lipid, and a PEG lipid comprising a compound having Formula XI. In some embodiments, a lipid nanoparticle comprises 49 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG. In some embodiments, a lipid nanoparticle comprises 49 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG. In some embodiments, a lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG. In some embodiments, an LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1. In some embodiments, an LNP of the present disclosure comprises an N:P ratio of about 6:1. In some embodiments, an LNP of the present disclosure comprises an N:P ratio of about 3:1, 4:1, or 5:1. In some embodiments, an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1. In some embodiments, an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1. In some embodiments, an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1. Some embodiments comprise a composition having one or more LNPs having a diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. Some embodiments comprise a composition having a mean LNP diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. In some embodiments, the composition has a mean LNP diameter from about 30nm to about 150nm, or a mean diameter from about 60nm to about 120nm. An LNP may comprise or one or more types of lipids, including but not limited to amino lipids (e.g., ionizable amino lipids), neutral lipids, non-cationic lipids, charged lipids, PEG- modified lipids, phospholipids, structural lipids and sterols. In some embodiments, the composition comprises a liposome. A liposome is a lipid particle comprising lipids arranged into one or more concentric lipid bilayers around a central region. The central region of a liposome may comprise an aqueous solution, suspension, or other aqueous composition. In some embodiments, a lipid nanoparticle may comprise two or more components (e.g., amino lipid and nucleic acid, PEG-lipid, phospholipid, structural lipid). For instance, a lipid nanoparticle may comprise an amino lipid and a nucleic acid. Compositions comprising lipid nanoparticles, such as those described herein, may be used for a wide variety of applications, including the stealth delivery of mRNA with minimal adverse innate immune response. Effective in vivo delivery of nucleic acids represents a continuing medical challenge. Exogenous nucleic acids (i.e., originating from outside of a cell or organism) are readily degraded in the body, e.g., by the immune system. Accordingly, effective delivery of nucleic acids to cells often requires the use of a particulate carrier (e.g., lipid nanoparticles). The particulate carrier should be formulated to have minimal particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids intracellularly, and illicit no or minimal immune response. To achieve minimal particle aggregation and pre-delivery stability, many conventional particulate carriers have relied on the presence and/or concentration of certain components (e.g., PEG-lipid). However, it has been discovered that certain components may decrease the stability of encapsulated nucleic acids (e.g., mRNA molecules). The reduced stability may limit the broad applicability of the particulate carriers. As such, there remains a need for methods by which to improve the stability of nucleic acid (e.g., mRNA) encapsulated within lipid nanoparticles. In some embodiments, lipid nanoparticles comprise one or more of ionizable molecules, polynucleotides, and optional components, such as structural lipids, sterols, neutral lipids, phospholipids and a molecule capable of reducing particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipid), such as those described above. In some embodiments, an LNP described herein may include one or more ionizable molecules (e.g., amino lipids or ionizable lipids). The ionizable molecule may comprise a charged group and may have a certain pKa. In certain embodiments, the pKa of the ionizable molecule may be greater than or equal to about 6, greater than or equal to about 6.2, greater than or equal to about 6.5, greater than or equal to about 6.8, greater than or equal to about 7, greater than or equal to about 7.2, greater than or equal to about 7.5, greater than or equal to about 7.8, greater than or equal to about 8. In some embodiments, the pKa of the ionizable molecule may be less than or equal to about 10, less than or equal to about 9.8, less than or equal to about 9.5, less than or equal to about 9.2, less than or equal to about 9.0, less than or equal to about 8.8, or less than or equal to about 8.5. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 6 and less than or equal to about 8.5). Other ranges are also possible. In embodiments in which more than one type of ionizable molecule are present in a particle, each type of ionizable molecule may independently have a pKa in one or more of the ranges described above. In general, an ionizable molecule comprises one or more charged groups. In some embodiments, an ionizable molecule may be positively charged or negatively charged. For instance, an ionizable molecule may be positively charged. For example, an ionizable molecule may comprise an amine group. As used herein, the term “ionizable molecule” has its ordinary meaning in the art and may refer to a molecule or matrix comprising one or more charged moiety. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule and/or matrix may be selected as desired. In some cases, an ionizable molecule (e.g., an amino lipid or ionizable lipid) may include one or more precursor moieties that can be converted to charged moieties. For instance, the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above. As a non-limiting specific example, the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively. Those of ordinary skill in the art will be able to determine whether a given chemical moiety carries a formal electronic charge (for example, by inspection, pH titration, ionic conductivity measurements, etc.), and/or whether a given chemical moiety can be reacted (e.g., hydrolyzed) to form a chemical moiety that carries a formal electronic charge. The ionizable molecule (e.g., amino lipid or ionizable lipid) may have any suitable molecular weight. In certain embodiments, the molecular weight of an ionizable molecule is less than or equal to about 2,500 g/mol, less than or equal to about 2,000 g/mol, less than or equal to about 1,500 g/mol, less than or equal to about 1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equal to about 900 g/mol, less than or equal to about 800 g/mol, less than or equal to about 700 g/mol, less than or equal to about 600 g/mol, less than or equal to about 500 g/mol, less than or equal to about 400 g/mol, less than or equal to about 300 g/mol, less than or equal to about 200 g/mol, or less than or equal to about 100 g/mol. In some instances, the molecular weight of an ionizable molecule is greater than or equal to about 100 g/mol, greater than or equal to about 200 g/mol, greater than or equal to about 300 g/mol, greater than or equal to about 400 g/mol, greater than or equal to about 500 g/mol, greater than or equal to about 600 g/mol, greater than or equal to about 700 g/mol, greater than or equal to about 1000 g/mol, greater than or equal to about 1,250 g/mol, greater than or equal to about 1,500 g/mol, greater than or equal to about 1,750 g/mol, greater than or equal to about 2,000 g/mol, or greater than or equal to about 2,250 g/mol. Combinations of the above ranges (e.g., at least about 200 g/mol and less than or equal to about 2,500 g/mol) are also possible. In embodiments in which more than one type of ionizable molecules are present in a particle, each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above. In some embodiments, the percentage (e.g., by weight, or by mole) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and/or of all the ionizable molecules within a particle may be greater than or equal to about 15%, greater than or equal to about 16%, greater than or equal to about 17%, greater than or equal to about 18%, greater than or equal to about 19%, greater than or equal to about 20%, greater than or equal to about 21%, greater than or equal to about 22%, greater than or equal to about 23%, greater than or equal to about 24%, greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 35%, greater than or equal to about 40%, greater than or equal to about 42%, greater than or equal to about 45%, greater than or equal to about 48%, greater than or equal to about 50%, greater than or equal to about 52%, greater than or equal to about 55%, greater than or equal to about 58%, greater than or equal to about 60%, greater than or equal to about 62%, greater than or equal to about 65%, or greater than or equal to about 68%. In some instances, the percentage (e.g., by weight, or by mole) may be less than or equal to about 70%, less than or equal to about 68%, less than or equal to about 65%, less than or equal to about 62%, less than or equal to about 60%, less than or equal to about 58%, less than or equal to about 55%, less than or equal to about 52%, less than or equal to about 50%, or less than or equal to about 48%. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 20% and less than or equal to about 60%, greater than or equal to 40% and less than or equal to about 55%, etc.). In embodiments in which more than one type of ionizable molecule is present in a particle, each type of ionizable molecule may independently have a percentage (e.g., by weight, or by mole) in one or more of the ranges described above. The percentage (e.g., by weight, or by mole) may be determined by extracting the ionizable molecule(s) from the dried particles using, e.g., organic solvents, and measuring the quantity of the agent using high pressure liquid chromatography (i.e., HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS). Those of ordinary skill in the art would be knowledgeable of techniques to determine the quantity of a component using the above-referenced techniques. For example, HPLC may be used to quantify the amount of a component, by, e.g., comparing the area under the curve of a HPLC chromatogram to a standard curve. It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given their ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way. Vaccine Compositions Some aspects of the present disclosure relate to compositions for use to induce an immune response in a subject (e.g., as vaccines against a flavivirus or multiple flaviviruses). In some embodiments, the flavivirus is selected from the group consisting of: Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus and Omsk virus. In one embodiment, the flavivirus is Dengue virus. In another embodiment, the flavivirus is Zika virus. In some embodiments, the flavivirus is Dengue virus and Zika virus. Multivalent Vaccines The compositions, as provided herein, may include single mRNA molecules or multiple RNAs (e.g., mRNAs) encoding two or more antigens of the same or different species. In some embodiments, composition includes an mRNA or multiple RNAs (e.g., mRNAs) encoding two or more flavivirus proteins. In some embodiments, the mRNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more flavivirus (e.g., DENV) proteins. In some embodiments, the composition comprises a first mRNA encoding a first stabilized heterotetramer and a second mRNA encoding a second stabilized heterotetramer. In some embodiments, the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the first and second stabilized heterotetramers are of the same DENV serotype. In some embodiments, the first and second stabilized heterotetramers are of different DENV serotypes. In some embodiments, the composition comprises a first mRNA encoding a first stabilized heterotetramer, a second mRNA encoding a second stabilized heterotetramer, and a third mRNA encoding a third stabilized heterotetramer. In some embodiments, the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the third stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the first, second, and third stabilized heterotetramers are of the same DENV serotype. In some embodiments, the first and second stabilized heterotetramers are of the same DENV serotype and the third stabilized heterotetramer is of a different DENV serotype. In some embodiments, the first, second, and third stabilized heterotetramers are of different DENV serotypes. In some embodiments, the composition comprises a first mRNA encoding a stabilized DENV1 heterotetramer, a second mRNA encoding a stabilized DENV2 heterotetramer, a third mRNA encoding a stabilized DENV3 heterotetramer, and a fourth mRNA encoding a stabilized DENV4 heterotetramer. In some embodiments, two or more different mRNA encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different mRNA encoding antigens may be formulated in separate lipid nanoparticles (each mRNA formulated in a single lipid nanoparticle). Lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNAs (e.g., mRNAs) encoding multiple antigens) or may be administered separately. Some aspects relate to multivalent vaccines that comprise components to protect a subject against more than one flavivirus. Multivalent flavivirus vaccines can include two (bivalent), three (trivalent), four (quadrivalent), five (pentavalent), or more components that each independently are designed to protect against one of a variety of flaviviruses. In some aspects, a multivalent vaccine comprises two mRNAs, each directed to a different serotype of a flavivirus (e.g., Dengue virus). In some embodiments, the mRNAs are present at a 1:1, 2:1, 4:1, 1:1:1, 2:1:1, 4:2:1, 1:1:1:1, 2:1:1:1, 4:2:1:1, or 4:2:2:1 mass ratio or a 1:1, 2:1, 4:1, 1:1:1, 2:1:1, 4:2:1, 1:1:1:1, 2:1:1:1, 4:2:1:1, or 4:2:2:1 molar ratio. Combination Vaccines Some embodiments of vaccines include combination vaccines. A "combination vaccine", as used herein, refers to a vaccine comprising two or more components for eliciting an immune response against multiple flaviviruses. In some embodiments, the combination vaccine elicits an immune response against Dengue virus and Zika virus (e.g., an immune response agains DENV1, DENV2, DENV3, DENV4, and/or ZIKV). In some embodiments, a combination vaccine includes one or more RNAs encoding an antigen of more than one flavivirus. For example, a combination vaccine may comprise mRNAs encoding stabilized E and M protein heterotetramers from DENV1, DENV2, DENV3, DENV4, and ZIKV. In some embodiments, the composition comprises a first mRNA encoding a first stabilized heterotetramer and a second mRNA encoding a second stabilized heterotetramer. In some embodiments, the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the second stabilized heterotetramer is a ZIKV stabilized heterotetramer. In some embodiments, the composition comprises a first mRNA encoding a first stabilized heterotetramer, a second mRNA encoding a second stabilized heterotetramer, and a third mRNA encoding a third stabilized heterotetramer. In some embodiments, the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the third stabilized heterotetramer is a ZIKV stabilized heterotetramer. In some embodiments, the first and second stabilized heterotetramers are of the same DENV serotype. In some embodiments, the first and second stabilized heterotetramers are of different DENV serotypes. In some embodiments, the composition comprises a first mRNA encoding a first stabilized heterotetramer, a second mRNA encoding a second stabilized heterotetramer, a third mRNA encoding a third stabilized heterotetramer, and fourth mRNA encoding a fourth stabilized heterotetramer. In some embodiments, the first stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the second stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the third stabilized heterotetramer is a DENV1, DENV2, DENV3, or DENV4 stabilized heterotetramer. In some embodiments, the fourth stabilized heterotetramer is a ZIKV stabilized heterotetramer. In some embodiments, the first, second, and third stabilized heterotetramers are of the same DENV serotype. In some embodiments, the first and second stabilized heterotetramers are of the same DENV serotype and the third stabilized heterotetramer is of a different DENV serotype. In some embodiments, the first, second, and third stabilized heterotetramers are of different DENV serotypes. In some embodiments, the composition comprises a first mRNA encoding a stabilized DENV1 heterotetramer, a second mRNA encoding a stabilized DENV2 heterotetramer, a third mRNA encoding a stabilized DENV3 heterotetramer, a fourth mRNA encoding a stabilized DENV4 heterotetramer, and a fifth mRNA encoding a stabilized ZIKV heterotetramer. In some embodiments, the mRNAs are present in various ratios in order to induce efficacious immune responses. In some embodiments, the ratio of mRNA encoding DENV to mRNA encoding ZIKV is a 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 molar or mass ratio. In some embodiments, the mRNA encoding the DENV serotype heterotetramers (e.g., DENV1, DENV2, DENV3, and DENV4) are present at a 1:1, 2:1, 4:1, 1:1:1, 2:1:1, 4:2:1, 1:1:1:1, 2:1:1:1, 4:2:1:1, or 4:2:2:1 molar or mass ratio. In some embodiments, the nucleotides encoding the stabilized heterotetramers are encapsulated in lipid nanoparticles for delivery. In some embodiments, one or more RNAs encoding polypeptides from different flaviviruses are encapsulated in a single lipid nanoparticle. In some embodiments, each RNA encoding a polypeptide from a single flavivirus is encapsulated in separate lipid nanoparticles. These nanoparticles can then be combined into a single vaccine composition or administered separately. Pharmaceutical Formulations Provided herein are compositions (e.g., pharmaceutical compositions, such as vaccines), methods, kits and reagents for prevention of flavivirus infections and other conditions directly or indirectly cause by flavivirus infection in humans and other mammals, for example. In some embodiments the compositions are protein vaccines, DNA vaccines or mRNA vaccines. In some embodiments, the compositions containing mRNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the mRNA are translated in vivo to produce an antigenic polypeptide (antigen). An “effective amount” of a composition (e.g., comprising mRNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the mRNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a composition provides an immune response as a function of antigen administration to a subject or production in the cells of the subject. In embodiments, where mRNA is administered, an effective amount of the composition containing mRNA having at least one chemical modification are more efficient than a composition containing a corresponding 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 mRNA vaccine), increased protein translation and/or expression 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. The term "pharmaceutical composition" refers to the combination of an active agent (e.g., mRNA) with a carrier (e.g., lipid composition, e.g., LNP)), inert or active, making the composition especially suitable for prophylactic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. In some embodiments, the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for the prevention of a flavivirus infection. A composition may be administered prophylactically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase. In some embodiments, the amount of mRNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis. A composition may be administered 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, 12 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, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more. In exemplary 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, or 6 months. As is described herein, the booster may comprise the same or different mRNA as compared to the earlier administration of the prophylactic composition. The booster, in some embodiments is monovalent (e.g., the mRNA encodes a single antigen). In some embodiments, the booster is multivalent (e.g., the mRNA encodes more than one antigen). In some embodiments, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful. In some embodiments, a composition disclosed herein is administered to a subject enterally. In some embodiments, an enteral administration of the composition is oral. In some embodiments, a composition disclosed herein is administered to the subject parenterally. In some embodiments, a composition disclosed herein is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs. In some embodiments, the administration is intramuscular. A composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. Provided herein are pharmaceutical compositions including mRNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients. The mRNA may be formulated or administered alone or in conjunction with one or more other components. For example, a vaccine may comprise other components including, but not limited to, adjuvants. In some embodiments, a vaccine does not include an adjuvant (it is adjuvant free). An mRNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccines comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccines, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In some embodiments, a vaccine is administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the mRNA contained therein, for example, mRNA encoding a stabilized heterotetramer. Formulations of the vaccines described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., 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. Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure 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 may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient. Methods of Prophylaxis (Induction of Immune Response) Provided herein are compositions (e.g., vaccines), methods, kits and reagents for inducing an immune response in a subject (e.g., an immune response to the composition) in humans and other mammals to flaviviruses. A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject. In some embodiments, a composition is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen-specific immune response. In some embodiments, the mRNA encoding the flavivirus protein is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject. Prophylactic protection from other conditions cause directly or indirectly by flavivirus infection can be achieved following administration of a composition of the present disclosure. The compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the composition once (optionally followed by a single booster). Dosing may need to be adjusted accordingly. A method of eliciting an immune response in a subject against a flavivirus protein (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, a method involves administering to the subject a vaccine comprising a mRNA having an open reading frame encoding a flavivirus protein (or multiple antigens), thereby inducing in the subject an immune response specific to the flavivirus protein (or multiple antigens), wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti- antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen. An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen. A prophylactically effective dose is an effective dose that prevents infection with the virus and/or reduces symptoms or negative outcomes from a viral infection at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine, the subject heterotetrameric vaccines are non-infectious and can be administered as proteins or nucleic acids (e.g., mRNAs). A composition of the present disclosure may be administered by any route that results in a prophylactically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. The present disclosure provides methods comprising administering the subject 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. In the case of mRNA, the effective amount of the mRNA (e.g., an effective dose), as provided herein, may be as low as 20 µg, administered for example as a single dose or as two 10 µg doses (e.g., a first effective vaccine dose and a second effective vaccine dose). In some embodiments, the first effective vaccine dose and the second effective vaccine dose are the same amount. In some embodiments, the first effective vaccine dose and the second effective vaccine dose are different amounts. In some embodiments, the effective amount is a total dose of 5 µg- 30 µg, 5 µg -25 µg, 5 µg -20 µg, 5 µg -15 µg, 5 µg -10 µg, 10 µg -30 µg, 10 µg -25 µg, 10 µg- 20 µg, 10 µg -15 µg, 15 µg -30 µg, 15 µg -25 µg, 15 µg -20 µg, 20 µg -30 µg, 25 µg -30 µg, or 25 µg-300 µg. In some embodiments, the effective dose (e.g., effective amount) is at least 10 µg and less than 25 µg of the composition. In some embodiments, the effective dose (e.g., effective amount) is at least 5 µg and less than 25 µg of the composition. For example, the effective amount may be a total dose of 5 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 35 µg, 40 µg, 45 µg, 50 µg, 55 µg, 60 µg, 65 µg, 70 µg, 75 µg, 80 µg, 85 µg, 90 µg, 95 µg, 100 µg, 110 µg, 120 µg, 130 µg, 140 µg, 150 µg, 160 µg, 170 µg, 180 µg, 190 µg, 200 µg, 250 µg, or 300 µg. In some embodiments, the effective amount (e.g., effective dose) is a total dose of 10 μg. In some embodiments, the effective amount is a total dose of 20 μg (e.g., two 10 μg doses). In some embodiments, the effective amount is a total dose of 25 μg. In some embodiments, the effective amount is a total dose of 30 μg. In some embodiments, the effective amount is a total dose of 50 μg. In some embodiments, the effective amount is a total dose of 60 μg (e.g., two 30 μg doses). In some embodiments, the effective amount is a total dose of 75 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a total dose of 150 μg. In some embodiments, the effective amount is a total dose of 200 μg. In some embodiments, the effective amount is a total dose of 250 μg. In some embodiments, the effective amount is a total dose of 300 μg. The mRNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). In some embodiments the composition or vaccine is administered to a subject intramuscularly. Vaccine Efficacy Some aspects of the present disclosure provide formulations of the compositions (e.g., RNA vaccines), wherein the mRNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a flavivirus antigen). “An effective amount” is a dose of the mRNA effective to produce an antigen-specific immune response. Also provided herein are methods of inducing an antigen-specific immune response in a subject. In some embodiments, the antigen-specific immune response is characterized by measuring an anti-DENV antigen antibody titer (e.g., neutralization titer) produced in a subject administered a composition as provided herein. In some embodiments, the antigen-specific immune response is characterized by measuring an anti-ZIKV antigen antibody titer (e.g., neutralization titer) produced in a subject administered a composition as provided herein. In some embodiments, the antigen-specific immune response is characterized by measuring an anti-YFV antigen antibody titer (e.g., neutralization titer) produced in a subject administered a composition 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 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. Other Embodiments Other embodiments of the present disclosure include: Embodiment 1. A messenger ribonucleic acid (mRNA) vaccine, comprising: an mRNA comprising an open reading frame encoding a modified dengue virus (DENV) protein, relative to a corresponding naturally occurring DENV protein; and a lipid nanoparticle. Embodiment 2. The mRNA vaccine of embodiment 1, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363. Embodiment 3. A messenger ribonucleic acid (mRNA) vaccine, comprising: an mRNA comprising an open reading frame encoding a Zika virus (ZIKV) protein, relative to a corresponding naturally occurring ZIKV protein; and a lipid nanoparticle. Embodiment 4. The mRNA vaccine of embodiment 3, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352. Embodiment 5. A messenger ribonucleic acid (mRNA) vaccine, comprising: an mRNA comprising an open reading frame encoding a Yellow fever virus (YFV) protein, relative to a corresponding naturally occurring YFV protein; and a lipid nanoparticle. Embodiment 6. The mRNA vaccine of embodiment 5, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214. Embodiment 7. A messenger ribonucleic acid (mRNA) vaccine, comprising: an mRNA comprising an open reading frame encoding a dengue virus (DENV) protein, relative to a corresponding naturally occurring DENV protein; an mRNA comprising an open reading frame encoding a Zika virus (ZIKV) protein, relative to a corresponding naturally occurring ZIKV protein; and a lipid nanoparticle. Embodiment 8. The mRNA vaccine of embodiment 7, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363. Embodiment 9. The mRNA vaccine of embodiment 7 or 8, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170-172, 177-178, 212-213, 285-301, and 347-352. Embodiment 10. The mRNA vaccine of any one of embodiments 6-9, further comprising an mRNA comprising an open reading frame encoding a Yellow fever virus (YFV) protein, relative to a corresponding naturally occurring YFV protein; and a lipid nanoparticle. Embodiment 11. The mRNA vaccine of embodiment 10, wherein the protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214. Embodiment 12. The mRNA vaccine of any one of the preceding embodiments, wherein the one or more mRNA(s) of the vaccine comprises a chemical modification. Embodiment 13. The mRNA vaccine of embodiment 12, wherein 100% of the uracil nucleotides of the one or more mRNA(s) comprise a chemical modification. Embodiment 14. The mRNA vaccine of embodiment 12 or 13, wherein the chemical modification is 1-methylpseudouracil. Embodiment 15. The mRNA vaccine of any one of the preceding embodiments, wherein the lipid nanoparticle comprises an ionizable lipid, a neutral lipid, a sterol, and a PEG-modified lipid. Embodiment 16. The mRNA vaccine of embodiment 15, wherein the lipid nanoparticle comprises 40–50 mol% ionizable lipid, 5–15 mol% neutral lipid, 30–50 mol% sterol, and 0.5–3 mol% PEG-modified lipid. Embodiment 17. The mRNA vaccine of embodiment 15 or 16, wherein the ionizable lipid comprises a structure of Compound (I):
Figure imgf000192_0001
the neutral lipid is distearoylphosphatidylcholine (DSPC); the sterol is cholesterol; and/or the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG-DMG). Embodiment 18. A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject one or more doses of the mRNA vaccine of any one of the preceding embodiments in an effective amount to produce an immune response to the protein. Embodiment 19. The method of embodiment 18, wherein the vaccine is administered intramuscularly. Embodiment 20. The method of embodiment 18 or 19 comprising administering a single dose of the vaccine to the subject. Embodiment 21. The method of embodiment 18 or 19 comprising administering a prime dose and a booster dose of the vaccine to the subject. Embodiment 22. A protein comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-21, 26-62, 67-107, 110-139, 143-148, 156-166, 173-176, 179-186, 189-211, 215-237, 246-255, 302-346, and 353-363. Embodiment 23. A protein comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 22-25, 63-66, 108-109, 140-142, 170- 172, 177-178, 212-213, 285-301, and 347-352. Embodiment 24. A protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 167-169, 238-245, 187-188, and 214. Embodiment 25. A fusion protein comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein: (a) the E protein comprises at least one substitution at one or more positions selected from: (i) T76, R93, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to SEQ ID NOs: 274, 276, or 280 ; or (ii) T76, K93, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A493, relative to SEQ ID NO: 278 ; or, (iii) T76, K93, L107, Q131, E133, T194, D197, S199, D200, H214, A248, T267, A270, A272, L273, S286, A319, H323, T325, R402, S403, K443, Q447, G450, A451, K454, S503, or A504, relative to SEQ ID NO: 282 ; and (b) the M protein comprises at least one substitution at one or more positions selected from: (i) S93, V94, A95, L96, A97, H99, V100, L102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or A167, relative to SEQ ID NO: 273 ; or (ii) S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to SEQ ID NO: 275 or 277 ; or (iii) S93, V94, A95, L96, T97, H99, S100, M102, L104, E105, T106, R107, A108, E109, S113, S114, A117, K119, Q122, E125, S126, R130, S165, Y166, or G167, relative to SEQ ID NO: 279 ; or (iv) A95, V96, T97, L98, P99, H101, S102, R104, L106, Q107, T108, R109, S110, Q111, E115, S116, Y119, K121, I124, E127, N128, R132, A167, Y168, or S169, relative to SEQ ID NO: 281. Embodiment 26. The fusion protein of embodiment 25, wherein the fusion protein comprises, from N-terminus to C-terminus, the E protein and the M protein. Embodiment 27. The fusion protein of embodiment 25, wherein the fusion protein comprises, from N-terminus to C-terminus, the M protein and the E protein. Embodiment 28. The fusion protein of any one of embodiments 25-27, further comprising a precursor (Pr) protein. Embodiment 29. The fusion protein of embodiment 28, wherein the fusion protein comprises, from N-terminus to C-terminus, the Pr protein, the M protein and the E protein. Embodiment 30. The fusion protein of any one of embodiments 25-29, further comprising a linker. Embodiment 31. The fusion protein of embodiment 30, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 149-155, and 364-365. Embodiment 32. The fusion protein of any one of embodiments 30-31, wherein the E protein and the M protein are linked by the linker. Embodiment 33. A messenger ribonucleic acid (mRNA) vaccine capable of inducing an immune response against a flavivirus antigen, wherein the mRNA vaccine comprises an mRNA polynucleotide having an open reading frame (ORF) encoding a fusion protein, wherein the fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 273-281 , and wherein the fusion protein comprises a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein: (a) the E protein comprises at least one substitution at one or more positions selected from: (i) T76, R93, L107, Q131, E133, T189, D192, N194, E195, H209, A243, T262, T265, A267, T268, A280, A313, H317, T319, K393, K394, K434, Q438, G441, T442, G445, Q494, or A495, relative to SEQ ID NOs: 274, 276 or 280 ; or (ii) T76, K93, L107, Q131, E133, T187, D190, N192, E193, H207, A241, T260, T263, A265, T266, A278, S311, H315, T317, K391, K392, K432, Q436, G439, S440, T443, Q492, or A493, relative to SEQ ID NO: 278 ; or (iii) T76, K93, L107, Q131, E133, T194, D197, S199, D200, H214, A248, T267, A270, A272, L273, S286, A319, H323, T325, R402, S403, K443, Q447, G450, A451, K454, S503, or A504, relative to SEQ ID NO: 282 ; and, (b) the M protein comprises at least one substitution at one or more positions selected from: (i) S93, V94, A95, L96, A97, H99, V100, L102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or A167, relative to SEQ ID NO: 273 ; or (ii) S93, V94, A95, L96, V97, H99, V100, M102, L104, E105, T106, R107, T108, E109, S113, S114, A117, K119, Q122, E125, T126, R130, S165, M166, or T167, relative to SEQ ID NO: 275 or 277 ; or (iii) S93, V94, A95, L96, T97, H99, S100, M102, L104, E105, T106, R107, A108, E109, S113, S114, A117, K119, Q122, E125, S126, R130, S165, Y166, or G167, relative to SEQ ID NO: 279 ; or (iv) A95, V96, T97, L98, P99, H101, S102, R104, L106, Q107, T108, R109, S110, Q111, E115, S116, Y119, K121, I124, E127, N128, R132, A167, Y168, or S169, relative to SEQ ID NO: 281. Embodiment 34. The mRNA vaccine of embodiment 33, wherein the fusion protein comprises, from N-terminus to C-terminus, the E protein and the M protein. Embodiment 35. The mRNA vaccine of embodiment 33, wherein the fusion protein comprises, from N-terminus to C-terminus, the M protein and the E protein. Embodiment 36. The mRNA vaccine of any one of embodiments 33-35, wherein the fusion protein further comprises a precursor (Pr) protein. Embodiment 37. The mRNA vaccine of embodiment 36, wherein the fusion protein comprises, from N-terminus to C-terminus, the Pr protein, the M protein and the E protein. Embodiment 38. The mRNA vaccine of any one of embodiments 33-37, wherein the fusion protein further comprises a linker. Embodiment 39. The mRNA vaccine of embodiment 38, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 149-155, and 364-365. Embodiment 40. The mRNA vaccine of any one of embodiments 38-39, wherein the E protein and the M protein are linked by the linker. Embodiment 41. The mRNA vaccine of any one of embodiments 33-40, wherein the fusion protein further comprises a signal peptide. Embodiment 42. The mRNA vaccine of embodiment 41, wherein the signal peptide comprises an amino acid sequence selected from SEQ ID NOs: 271 or 272. Embodiment 43. The mRNA vaccine of any one of embodiments 33-42, wherein the flavivirus is selected from the group consisting of: Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus and Omsk virus. Embodiment 44. The mRNA vaccine of embodiment 43, wherein the flavivirus is Dengue virus. Embodiment 45. The mRNA vaccine of embodiment 43, wherein the flavivirus is Zika virus. Embodiment 46. A method of inducing neutralizing antibodies in a subject by administering a stabilized flavivirus heterotetramer comprising: (a) means for inducing an immune response to the flavivirus in the subject; and (b) a flavivirus envelope (E) protein, wherein the E protein comprises an amino acid substitution at each of position 76 and at position 107, wherein the positions are numbered by alignment of the E protein to SEQ ID NO: 276; and a flavivirus membrane (M) protein, wherein administration of the flavivirus heterotetramer has increased neutralization antibody titers compared to wild-type flavivirus prME protein. Embodiment 47. A stabilized flavivirus heterotetramer, the heterotetramer comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein the E protein comprises a fusion loop, a bc loop, and a transmembrane region; wherein the fusion loop comprises an amino acid substitution at position 107 and the bc loop comprises an amino acid substitution at position 76 and wherein the positions correspond to SEQ ID NO: 276; and the M protein comprises a N-terminus and a C-terminus; wherein the N-terminus comprises an N- terminal region comprising at least one amino acid substitution relative to the corresponding wild-type flavivirus M protein. EXAMPLES Example 1 – In Silico Expression of Selected Dengue, Zika, and Yellow Fever Antigens Virus-like particles (VLPs) comprising various designed immunogens derived from ZIKV, DENV1, DENV2, DENV3, DENV4, and YFV were tested for their expression as plasmid DNA in Expi-293 cells. Prior work has demonstrated that using plasmid DNA is an appropriate surrogate for proteins expressed as mRNA. Each of the immunogens tested comprised either: (i) a Pr protein sequence followed by a membrane (M) protein and followed by an envelope (E) protein (“PrM/E”); (ii) a protein comprising an E protein followed by an M protein (“E/M”); or (iii) a protein comprising an M protein followed by an E protein (“M/E”). VLPs were designed and engineered based on visual inspection of the proteins using graphical software (e.g., chimeraX), and mutations were designed accordingly. pcDNA3.4 plasmids containing the gene sequence for the engineered VLPs were procured from GenScript. To test the expression of DENV2 designed immunogens, dot-blot expression profiles were performed on processed supernatant collected from Expi-293 cells transfected with engineered VLP plasmids. Expression was assessed using either EDE2 A11 (“A11”), a conformational broadly neutralizing antibody (bnAb), or 4G2, a fusion loop-binding, enhancing monoclonal antibody (mAb) (Table 5). In Table 5 below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale: “-” indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Wild-type DENV2 PrM/E and DENV2 M/E VLPs were used as controls (“WT” and “wt”, respectively). Analysis of dot-blot results showed design numbers DENV2-E/M_55, DENV2-E/M_56, DENV2-E/M_57, DENV2-M/E_111, DENV2-M/E_131, DENV2-M/E_133, DENV2-M/E_134, DENV2-M/E_135, and DENV2-M/E_136 displayed significantly higher expression than the WT VLPs when probed with the conformational bnAb which detects the desired E dimer epitope of DENV2. Conversely, when detecting expression using the 4G2 antibody, all designs showed lower detection than WT VLPs.4G2 can lead to the enhancement of infection rather than protection, and thus lower detection with this antibody indicates that the epitope region for this class of antibodies was successfully averted without compromising the binding to protective antibodies such as A11E. Table 5. DENV2 VLPs – Relative Expression Data Note: EDE2 A11, specific for DENVs1-4; FLE 4G2, anti-fusion loop antibody. Detection levels are shown relative to the expression of WT sample. Detection signal is dependent on the expression level as well as the affinity of a given antibody, and thus each detection should be compared for designs within a virus type and for one particular antibody. For example, the table below shows that DENV2-M/E_136 gave highest detection with the A11 antibody compared to detection of WT with the same antibody. In the table,
Figure imgf000197_0001
indicates no detectable signal, which does not indicate lack of expression. For example, no signal was observed for DENV2-prME_AS4 with the 4G2 antibody compared to the background, but a signal was observed with the A11 antibody, indicating that this design preferred binding to A11 and has no detectable binding to 4G2.
Figure imgf000197_0002
Figure imgf000198_0001
Figure imgf000199_0001
To test the expression of ZIKV, DENV1, DENV3, and DENV4 designed immunogens, dot-blot expression profiles were performed as described above. Expression was assessed using an A11 bnAb, a 4G2 mAb, or an EDE1 C8 bnAb (Table 6). In Table 6 below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale: “-” indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Because A11 does not bind well to ZIKV, low signal levels were observed. Conversely, DENV3 designs 1-4 and 50 showed bright signals, indicating higher expression than the WT VLP. The signals of DENV1 and DENV4 designed VLPs were low when measured with the A11 bnAb. Conversely, the anti-fusion loop 4G2 mAb recognized the WT VLPs and DENV3 design 1, indicating that this particular design was not desirable. EDE1 C8 bnAb (“C8”) was used to detect ZIKV VLP designs. Dot-blot analysis using the C8 bnAb demonstrated bright signals in all ZIKV VLP designs except 2-4, indicating recognition by a desired conformational antibody. Designs showing high reactivity to the broadly neutralizing antibodies and low reactivity to the anti-fusion loop antibodies were selected for further analysis. Table 6. ZIKV, DENV1, DENV3 and DENV4 – Relative Expression Data Note: EDE1 C8, specific for DENVs1-4 and ZIKV; EDE2 A11, specific for DENVs1-4; FLE 4G2, anti-fusion loop antibody. Detection levels are comparable only within each virus, and are indicated as relative to their WT version. For example, for ZIKV, ZV-WT is the reference. Also, even though Abs are cross-reactive their affinity for different viruses vary and thus can lead to high or low signal even if their expression is comparable. Signal when comparable to the background is indicated by “-“. Blank doesn’t mean there is no expression, but rather indicates that, compared to the highest signal in the blot, the expression detection signal for that particular construct was very low. For example, the 4G2 signal for ZIKV- prME_AS2 was close to the background level, but there is expression for this construct as detected by using the A11 antibody.
Figure imgf000200_0001
Expression of ZIKV, DENV1, DENV3, and DENV4 designed immunogens were further tested using a panel of conformational bnAbs, neutralizing antibodies, and non-neutralizing, enhancing mAbs (Tables 3A, 3B, and 3C, respectively). In the tables below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale: “-” indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. All bnAbs, cross-reactive antibodies, and serotype-specific antibodies showed higher expression of designed VLPs compared to WT VLPs, whereas the WT VLPs showed higher signal when detection was performed using the anti-fusion loop antibodies 4G2, ZVFL_5JHL and ZVFL_Z6 (undesired; Table 7C). These data demonstrated that the engineered VLPs show higher expression when recognized by a desired conformational antibody. Table 7A. ZIKV, DENV1, DENV3 and DENV4 – Relative Expression Data Note: C10, specific for DENVs1-4 and ZIKV; C8, specific for DENVs1-4 and ZIKV; B7, specific for DENVs1-4; A11, specific for DENVs1-4; SIgN_3C, specific for ZIKV, DENV2, DENV3; MZ4, specific for ZIKV, DENV2, DENV3; ZVDV1IDIII_Z021, specific for ZIKV and DENV1.
Figure imgf000201_0001
Figure imgf000202_0001
Table 7B. ZIKV, DENV1, DENV3 and DENV4 – Relative Expression Data (neutralizing antibodies) Note: DV1EDI_1F4, DENV1-specific; ZIKV_195, ZIKV-specific; ZVED_8DV6, ZIKV-specific; 2D22, DENV2-specific; DV2EIII_2C8, DENV2-specific; DV3dim_5J7, DENV3-specific; DV4EDI_5H2, DENV4-specific
Figure imgf000202_0002
Figure imgf000203_0001
Table 7C. ZIKV, DENV1, DENV3 and DENV4 – Relative Expression Data (non- neutralizing enhancing monoclonal antibodies)
Figure imgf000203_0002
Figure imgf000204_0002
VLPs showing the highest expression levels as measured by dot-blot analysis were chosen for electron microscopy analysis. The candidates included DENV152 (DENV1_52_E/M), DENV2102 (DENV2_M/E_102), DENV354 (DENV3_54_E/M VLP), and ZIKV 50 (ZIKV_50_E/M). Also tested were DENV2-M/E_102 VLPs in complex with Fab fragments of bnAb C8 (DENV2102 + Fab C8) and ZIKV_50_E/M VLPs in complex with Fab fragments of bnAb C8 (ZIKV 50 + Fab C8). Presence of the indicated VLPs was demonstrated by negative stain electron microscopy and cryo-EM (FIG.2; boxes show the VLPs). VLPs of sizes 40 to 60 nm were observed for each construct. Fab C8 binding was observed for DENV2 102 VLPs as protrusions on the smooth surface, while ZIKV 50 VLPs showed binding to Fab C8 in native conditions as observed with cryo-EM. To test the expression of YFV antigens, dot-blot expression profiles were performed as described above. Expression was assessed using either specific mAbs 5A (“5A”), or a YD73 mAb, which targets the domain III region of the YFV-E protein (EDIII) (Table 8). In the table below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale:
Figure imgf000204_0001
indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Brazilian and Asibi strains of YFV were tested. Bright signals were observed for both the Y50 and Y51 designs of the Brazilian strain when detection was performed using either antibody (Table 8), indicating enhanced expression of these YFV designed VLPs compared to the WT VLPs. Table 8. YFV – Relative Expression Data Note: 5A, monoclonal antibody against YFV; YD73, anti-EDIII antibody (targets domain III region); tested in duplicate (“1” and “2” in the table below); Brazilian and Asibi strains tested (“Br” and “Asb”, respectively)
Figure imgf000205_0002
Further ZIKV and DENV antigens were screened for expression using conformational and linear epitope antibodies (Table 9). In the table below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale:
Figure imgf000205_0001
indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Expression was measured by dot-blot analysis as described above. The cross-reactive antibodies used (i.e., EDE1 C10, EDE1 C8, EDE2 A11, SIgN-3C, and 4G2) have different binding affinities to all five viruses, thus their signal is used to discriminate expression of designs for the same virus. D1-005, D2-029, D3-002, D4-003, and Z50HANg demonstrated the highest expression and detection among the designs for the five different viruses. Table 9. ZIKV and DENV – Relative Expression Data Note: EDE1 C10, binds to ZIKV and DENVs1-4; EDE1 C8, binds to ZIKV and DENVs1-4; EDE2 A11, binds to DENVs1-4; DV4 EDI 5H2, binds to DENV4; SIgN-3C, binds to ZIKV and DENVs1-4; DV3_5J7, binds to DENV3; 2D22, binds to DENV2; 4G2, anti-fusion loop antibody
Figure imgf000205_0003
Figure imgf000206_0001
Figure imgf000207_0001
VLPs showing the highest expression levels as measured by dot-blot analysis were chosen for purification and further protein analysis. FIG.3 demonstrates the results of one of the candidates, ZIKV_HA_50_E/M VLPs, which were expressed in Expi293 cells then purified as described in “Methods” below. Further purification was then performed using streptavidin- tagged EDE1 Fab C8 that bound to the VLPs. The antibody-VLP complexes were captured by a streptavidin column, and VLPs were then eluted. These purified VLPs were then tested using SDS-PAGE in either non-reducing (-BME) or reducing (+BME) conditions (FIG.3, columns 2 and 3, respectively). The ZIKV_HA_50_E/M VLPs contain an engineered disulfide bond that stabilizes an (EM)2 heterotetramer, which is reduced in the presence of BME. FIG.3 demonstrates the presence of intact ZIKV_HA_50_E/M heterotetramers at ~130 kDa in non- reducing conditions (column 2) and heterodimers at ~65 kDa in reducing conditions (column 3), verifying the presence of a ZIKV_HA_50_E/M heterotetrameric complex containing the engineered disulfide bond. Additional ZIKV, DENV2, and DENV3 antigens were screened for expression using conformational and linear epitope antibodies (Table 10). In the table below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale: ” indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Expression was measured by dot-blot analysis as described above. Conformational binding antibodies EDE1 C8, EDE1 C10, EDE2 A11 were used for detection, as well as DENV3-specific antibody 5J7, ZIKV-specific antibody Z-195, and the fusion loop binding, enhancing antibody FLE Z6. Table 10. ZIKV, DENV2, and DENV3 – Relative Expression Data Note: EDE1 C10, binds to ZIKV and DENVs1-4; EDE1 C8, binds to ZIKV and DENVs1-4; EDE2 A11, binds to DENVs1-4; D3-5J7, binds DENV3; Z-195, binds ZIKV; and FLE Z6 is a fusion loop binding, enhancing antibody
Figure imgf000208_0001
Further ZIKV and DENV antigens were screened for expression using conformational and linear epitope antibodies (Table 11A-11D). In the tables below, dot-plot results were reviewed and then relative expression, depending on brightness was converted to the following scale: “-” indicates no detectable signal; and “+” to “++++” represent increasingly stronger signals relative to the wild-type sample. Expression was measured by dot-blot analysis as described above. The cross-reactive antibodies used to screen DENV1 and DENV2 designs (i.e., EDE1 C10, EDE2 A11, SIgN-3C, 1F4 DV1, and 4G2) have different binding affinities to all five viruses, thus their signal is used to discriminate expression of designs for the same virus. DENV3 and DENV4 designs were tested with other cross-reactive antibodies (e.g., EDE1 C8, EDE2 B7, 5JZ DV3, 5H2 DV4, and FLE Z6). Overall, D1-V2, D2-V2, D3-V2, and D4-V2 were selected for continued screening. Each design showed expression levels above that of wild-type and earlier version of the antigens, in addition to having less reactivity to the fusion loop antibodies (FLE 4G2 and FLE Z6). Table 11A. DENV1 and DENV2 – Relative Expression Data Note: EDE1 C10, binds to ZIKV and DENVs1-4; EDE2 A11, binds to DENVs1-4; D3- 5J7, binds DENV3; sIgN-3C, binds to ZIKV and DENVs1-4; DV1EDI_1F4, DENV1-specific and FLE Z6 is a fusion loop binding, enhancing antibody
Figure imgf000209_0001
Table 11B. DENV3 and DENV4 – Relative Expression Data Note: EDE1 C8, specific for DENVs1-4 and ZIKV; EDE2 B7, specific for DENVs1-4; DV3 5J7, DENV3-specific; DV45H2, DENV4-specific; and FLE Z6 is a fusion loop binding, enhancing antibody
Figure imgf000209_0002
Figure imgf000210_0001
Table 11C. DENV1-4 – Relative Expression Data (Broadly Neutralizing Antibodies) Note: EDE1 C8, specific for DENVs1-4 and ZIKV; EDE1 C10, binds to ZIKV and DENVs1-4; EDE2 A11, binds to DENVs1-4; EDE2 B7, specific for DENVs1-4; sIgN-3C, binds to ZIKV and DENVs1-4; and MZ4, specific for ZIKV, DENV2, DENV3
Figure imgf000210_0002
Figure imgf000211_0001
Table 11D. DENV1-4 – Relative Expression Data (Serotype-Specific Antibodies; Anti- Fusion Loop Antibodies) Note: 1F4 DV1, DENV1-specific antibody; 2J7 DV3, DENV3-specific antibody; 5H2 DV4, DENV4-specific antibody; FLE 4G2, anti-fusion loop antibody; FLE Z6, anti-fusion loop antibody
Figure imgf000211_0002
The expression selected antigens were further compared to their respective wild-type counterparts. Mass spectrometry (MS) was used to verify the higher expression of selected candidates compared to their respective wild-type (WT) prM/E proteins. In this assay, the relative abundance of the common peptide sequences between WT and the designed antigens (designed VLPs) was assessed. The top panel shows the comparison of intracellular and surface proteins in designed and WT antigens (FIG.4A). Cells expressing these VLPs were pelleted down and washed with VLPs before MS inspection of digested peptides. In each case, except for DENV2, the design antigens showed higher overall expression. FIG.4B shows the expression of soluble fractions (VLPs), and in each case, a higher abundance of designed antigens was observed. In the graphs, the wild-type proteins are as follows: WG D1 is DENV1 WT pr/ME; WT D2 is DENV2 WT pr/ME; WT 3 is DENV3 WT pr/ME; WT D4 is DENV4 WT pr/ME, and WT D1-D4 is a co-transfection of the four wild-type proteins (each at ¼ the amount used for the individual transfections). The four antigens tested were as follows: AS D1 is DV1_008_E/M; AS D2 is DV2-029_E/M; AS D3 is DENV3_HA_60_E/M; AS D4 is DV4-Ind_005_EM; and AS D1-4 is a co-transfection of all four proteins (each at ¼ the amount used for the individual transfections). The four selected antigens were then screened using negative stain electron microscopy (NSEM). NSEM was used to verify the presence of VLPs in a partially purified sample. In each case, VLPs corresponding to a size of ~50 Å were observed. Further VLPs appear to have a smooth appearance indicating prefusion state of the proteins on surface (FIG.5; boxes show the VLPs). Next, the four selected antigens were chosen for purification and further protein analysis. SDS-PAGE was used to validate the designed antigens. FIG.6 demonstrates the results of these candidates, which were expressed in Expi293 cells then purified as described in “Methods” below. Further purification was then performed using streptavidin-tagged EDE1 Fab C8 that bound to the VLPs. The antibody-VLP complexes were captured by a streptavidin column, and VLPs were then eluted. These purified VLPs were then tested using SDS-PAGE in either non- reducing (-BME) or reducing (+BME) conditions (FIG.6, left and right columns for each antigen, respectively). The DENV VLPs contain an engineered disulfide bond that stabilizes an (EM)2 heterotetramer, which is reduced in the presence of BME. FIG.6 demonstrates the presence of intact DENV E/M heterotetramers at ~122 kDa in non-reducing conditions (left column for each antigen) and heterodimers at ~61 kDa in reducing conditions (right column for each antigen), verifying the presence of DENV E/M heterotetrameric complexes containing the engineered disulfide bond and indicating that the protein is properly folded. Then, the selected DENV antigens were screened for expression using conformational and linear epitope antibodies (Table 12). Expression was measured by dot-blot analysis as described above. No binding was observed for the EDE1 GL C10 and DED1 GL C8 antibodies. In each instance, the mature EDE1 and EDE2 monoclonal antibodies were able to detect the designed antigens with higher affinity over their WT counterparts. Only germline EDE2 A11 showed specific signal of binding for the DENV1, -2 and -3 antigens and was unable to bind the DENV4 antigen. EDE1 C8 and C10 didn’t show specific binding signal to the WT or engineered V2 VLPs. Mature monoclonal antibodies showed binding to all four virus serotype antigens, but only germline EDE2 A11 was able bind to all four serotypes. EDE1 C8 and EDE1 C10 did not demonstrate complete binding, indicating that their affinities are higher than the highest tested concentration (400 nM). These results indicate that the engineered VLPs can engage the germline broadly neutralizing antibodies and monoclonal antibodies. Table 12 – Expression Data of Selected DENV Antigens
Figure imgf000213_0001
Next, antibody binding was tested using Fluorescence Activated Cell Sorting (FACS). Briefly, cells were transfected with the WT or designed antigens alone or in a tetravalent combination (all four DENV serotypes). The following WT VLPs were used: DENV1 wild type prM/E VLP, DENV2 wild type prM/E VLP, DENV3 wild type prM/E VLP, and DENV4 wild type prM/E VLP. In FIGs.7A-7D, “All 4” indicates the tetravalent co-transfection, and “Mock” represents untransfected cells. The designed antigens are indicated as “Stab” and are shown in light gray in FIGs.7A-7D. The designed antigens used were: DV1_008_E/M, DV2-029_E/M, DENV3_HA_60_E/M, and DV4-Ind_005_EM. The cells were pelleted and washed using PBS. Cell surface expression was probed using: serotype specific monoclonal antibodies, 1F4, 2D22, 5J7, and 5H2 (FIG.7A); anti-fusion loop monoclonal antibodies, 24211, 24220, and Z6 (FIG. 7B); broadly neutralizing antibodies, A11, B7, C8, C10 and SlgN3C (FIG.7C); and germline versions of the monoclonal antibodies, G-A11 and G-C10 (FIG.7D). In each case, the designed antigens gave higher signals than the WT, except for the undesired anti-fusion loop monoclonal antibodies (FIG.7B). Methods DOT-BLOT Analysis Various monoclonal antibodies (mAbs) were used to assess the expression of engineered VLPs in mammalian cells. Plasmids containing engineered VLPs were transfected into 1 mL Expi293 cells, and cell supernatant was harvested 3 days post-transfection. Cell supernatant was then filtered using a 0.1 µm filter.1.5 µL of the filtered (“clarified”) cell supernatant was applied to nitrocellulose membrane and probed using primary Dengue/Zika- or Flavavirus- specific antibodies of human or mouse origin. An anti-human or anti-mouse secondary antibody conjugated to a fluorescent tag was used for final detection, and readout was observed using a ChemiDoc imaging system. Negative Stain Electron Microscopy Engineered VLP candidates showing desired expression were selected for large scale production and structural studies using negative stain electron microscopy. Briefly, 200-500 mL Expi-293 cells were transfected, and cell supernatant was harvested 3 days post-transfection. Cell supernatant was mixed with polyethylene glycol 8000 (PEG8000) and centrifuged to concentrate and pellet VLPs; excess liquid was removed; and the pellet was then resuspended in 50 mM Tris (pH 8.0), 400 mM NaCl and 0.1 mM EDTA. The resuspended pellets were filtered through a 0.45 µm filter. The filtered suspension was further purified using a sucrose gradient of 5%, 10%, 15%, 20% and 25%, and the presence of VLPs was analyzed using a DOT-BLOT assay (see above). Fractions showing the presence of VLPs were pooled and further concentrated using 20% sucrose cushion centrifugation. Pelleted VLPs were resuspended in 500 µL of 50 mM Tris (pH 8.0), 400 mM NaCl. The resuspended VLPs were then passed through a CaptoCore 700 column (Cytiva) for clarification. The VLPs in the flowthrough were used for negative stain electron microscopy studies. For cryo-EM imaging, 3 µL of the resuspended VLPs were applied to the CaptoCore 700 column, washed with water followed by 3 washes with uranyl acetate. Cryo-EM grids were visualized using Tundra microscope at 69 K magnification. Example 2 – In Vitro Analysis of Designed Antigens Antibody binding was tested using Fluorescence Activated Cell Sorting (FACS). Briefly, cells were transfected with the WT or designed antigens alone or in a tetravalent combination (all four DENV serotypes). The following WT VLPs were used: DENV1 wild type prM/E VLP, DENV2 wild type prM/E VLP, DENV3 wild type prM/E VLP, and DENV4 wild type prM/E VLP. In FIGs.7A-7D, “All 4” indicates the tetravalent co-transfection, and “Mock” represents untransfected cells. The designed antigens are indicated as “Stab” and are shown in light gray in FIGs.7A-7D. The designed antigens used were: DV1_008_E/M, DV2-029_E/M, DENV3_HA_60_E/M, and DV4-Ind_005_EM. The cells were pelleted and washed using PBS. Cell surface expression was probed using the following antibodies: serotype specific monoclonal antibodies, 1F4, 2D22, 5J7, and 5H2 (FIG.7A); anti-fusion loop monoclonal antibodies, 24211, 24220, and Z6 (FIG.7B); broadly neutralizing antibodies, A11, B7, C8, C10 and SlgN3C (FIG. 7C); and germline versions of the monoclonal antibodies, G-A11 and G-C10 (FIG.7D). In each case, the designed antigens gave higher signals than the WT, except for the undesired anti-fusion loop monoclonal antibodies (FIG.7B). Example 3 – In Vivo Analysis of Selected Antigens The immunogenicity of selected Dengue virus (DENV) designed antigens was tested in vivo. Briefly, BALB/c mice were administered a prime dose of the selected composition on day 1 and a boost dose of the same composition of day 22. On days 21 and 57, serum samples were taken and analyzed. The monovalent DENV1, DENV2, DENV3, and DENV4 compositions were tested at two total dose levels: 1 μg and 4 μg. The compositions tested are shown in Table 13 below. Table 13 – Experimental Groups
Figure imgf000215_0001
* for the tetravalent compositions, a 1:1:1:1 ratio was used Using the serum samples collected, neutralization titers (NT50) were measured. Monovalent homologous antibody responses were determined and demonstrated that the designed antigens induce serotype-specific antibody responses to a significantly higher level than the control (PBS) (FIG.8). In particular, the DENV1 and DENV3 designs induced similar amounts of type-specific antibodies compared to their respective wild-type prME proteins; the DENV2 design induces more type-specific antibodies that its wild-type prME protein; and the DENV4 design induced less type-specific antibodies than its wild-type prME protein. Next, cross-reactivity was examined for each serotype. As is shown in FIG.9A, the engineered DENV1 designs induce cross-reactive antibody responses, and have a different pattern of cross-reactivity than the native VLPs. The results are represented in Table 14 below. Table 14 – Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
Figure imgf000215_0002
For DENV2, the results are depicted in FIG.9B and demonstrate that the engineered DENV2 designs induce cross-reactive antibody responses, although they induce fewer cross- reactive antibodies than the native VLPs. The results are represented in Table 15 below. Table 15 – Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
Figure imgf000216_0001
For DENV3, the results are depicted in FIG.9C and demonstrate that the engineered DENV3 designs induce cross-reactive antibody responses, although they induce fewer cross- reactive antibodies than the native VLPs. The results are represented in Table 16 below. Table 16 – Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
Figure imgf000216_0002
For DENV4, the results are depicted in FIG.9D and demonstrate that the engineered DENV4 designs induce cross-reactive antibody responses, although they, as well as the wild- type prME, induce the fewest cross-reactive antibodies of all the serotypes tested. The results are represented in Table 17 below. Table 17 – Cross-Reactivity Ratio (heterologous NT50/homologous NT50)
Figure imgf000216_0003
The tetravalent data was examined further. When a combination of all four antigens was administered (a tetravalent composition) it was found that there is no immunological interference with respect to DENV1, DENV2, and DENV4, as measured by neutralization titer; however, immunological interference was observed for DENV3 (FIG.10). In addition, the immunological balance across different DENV serotypes was examined, and neutralization titers were observed for each (FIG.11).

Claims

CLAIMS What is claimed is: 1. A stabilized flavivirus heterotetramer, the heterotetramer comprising a flavivirus envelope (E) protein and a flavivirus membrane (M) protein, wherein (a) the E protein comprises an amino acid substitution at each of two positions, wherein the two positions correspond with positions 76 and 107 of SEQ ID NO: 276, (b) the M protein comprises a N-terminus and a C-terminus; wherein the N-terminus comprises an N-terminal region comprising at least one amino acid substitution relative to a corresponding wild-type flavivirus M protein.
2. The stabilized flavivirus heterotetramer of claim 1, wherein the amino acid substitution at position 107 comprises a substitution with a charged bulky residue.
3. The stabilized flavivirus heterotetramer of claim 1 or 2, wherein the amino acid substitution at position 76 comprises a substitution with a charged bulky residue.
4. The stabilized flavivirus heterotetramer of claim 2 or 3, wherein the charged bulky residue is selected from the group consisting of: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), lysine (K), tyrosine (Y), and arginine (R).
5. The stabilized flavivirus heterotetramer of any one of claims 1-4, wherein the at least one substitution within the N-terminal region is selected from the following positions: 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105; wherein the positions correspond with positions 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105 of SEQ ID NO: 275.
6. The stabilized flavivirus heterotetramer of claim 5, wherein the at least one substitution within the N-terminal region is selected from the following substitutions: (a) the amino acid substitution at position 90 is valine (V) or isoleucine (I); (b) the amino acid substitution at position 91 is isoleucine (I) or cysteine (C); (c) the amino acid substitution at position 93 is isoleucine (I), phenylalanine (F), tyrosine (Y), or tryptophan (W); (d) the amino acid substitution at position 94 is isoleucine (I), leucine (L), asparagine (N), or lysine (K); (e) the amino acid substitution at position 96 is cysteine (C); (f) the amino acid substitution at position 97 is isoleucine (I) or cysteine (C); (g) the amino acid substitution at position 99 is isoleucine (I) or leucine (L); (h) the amino acid substitution at position 101 is methionine (M), isoleucine (I), or phenylalanine (F) (i) the amino acid substitution at position 103 is glycine (G), alanine (A), serine (S), or cysteine (C); (j) the amino acid substitution at position 104 is serine (S); and (k) the amino acid substitution at position 105 is cysteine (C); wherein the positions correspond with positions 90, 91, 93, 94, 96, 97, 99, 101, 103, 104, and 105 of SEQ ID NO: 275.
7. The stabilized flavivirus heterotetramer of any one of claims 1-6, wherein the E protein further comprises at least one additional substitution selected from the group consisting of: an amino acid substitution at position 131; an amino acid substitution at position 194; and an amino acid substitution at position 134; wherein the positions correspond with positions 131, 194, and 134 of SEQ ID NO: 276.
8. The stabilized flavivirus heterotetramer of claim 7, wherein at least one additional substitution is selected from the following amino acid substitutions: (a) the amino acid substitution at position 131 is alanine (A), asparagine (N), or leucine (L); (b) the amino acid substitution at position 194 is aspartic acid (D); and (c) the amino acid substitution at position 134 was asparagine (N) or aspartic acid (D); wherein the positions correspond with positions 131, 194, and 134 of SEQ ID NO: 276.
9. The stabilized flavivirus heterotetramer of any one of claims 1-8, wherein the E protein and the M protein comprise a fusion protein, the fusion protein comprising, from N-terminus to C-terminus, the M protein and the E protein.
10. The stabilized flavivirus heterotetramer of any one of claims 1-8, wherein the E protein and the M protein comprise a fusion protein, the fusion protein comprising, from N-terminus to C-terminus, the E protein and the M protein and optionally wherein a linker is positioned between the E protein and the M protein, further optionally wherein the linker is a flexible linker.
11. The stabilized flavivirus heterotetramer of claim 10, wherein the linker comprises GGGG (SEQ ID NO: 364) or GGPG (SEQ ID NO: 365).
12. The stabilized flavivirus heterotetramer of claim 10 or 11, wherein the E protein further comprises an amino acid substitution at position 495, wherein the position corresponds with position 495 if SEQ ID NO: 276; and/or the M protein further comprises an amino acid substitution at position 90, wherein the position corresponds to position 90 of SEQ ID NO: 275.
13. The stabilized flavivirus heterotetramer of claim 12, wherein the amino acid substitution of the E protein is a glycine (G) at position 495, wherein the position corresponds with position 495 of SEQ ID NO: 276; and/or wherein the amino acid substitution of the M protein is a glycine (G) at position 90, wherein the position corresponds to position 90 of SEQ ID NO: 275.
14. The stabilized flavivirus heterotetramer of any one of claims 10-13, further comprising a cavity created by (a) amino acid substitutions at positions 262 and 265 of the E protein, wherein the positions correspond with positions 262 and 265 of SEQ ID NO: 276; and (b) amino acid substitutions at position 97 of the M protein, wherein the position corresponds with position 97 of SEQ ID NO: 275.
15. The stabilized flavivirus heterotetramer of claim 14, wherein the amino acid substitutions of positions 262 and 265 of the E protein are each an alanine (A) or serine (S), wherein the positions correspond with positions 262 and 265 of SEQ ID NO: 276; and the amino acid substitution of position 97 of the M protein is alanine (A), wherein the position corresponds to position 97 of SEQ ID NO: 275.
16. The stabilized flavivirus heterotetramer of any one of claims 1-15 , wherein the E protein further comprises the following amino acid substitutions: a tyrosine (Y), glutamine (Q), or asparagine (N) at position 267; and a lysine (K) or arginine (R) at position 445; and wherein the positions correspond to positions 267 and 445 of SEQ ID NO: 276.
17. The stabilized flavivirus heterotetramer of any one of claims 1-16, wherein the N- terminal region of the M protein comprises at least one amino acid substitution of a non-cysteine amino acid with a cysteine amino acid, relative to the corresponding wild-type flavivirus M protein.
18. The stabilized flavivirus heterotetramer of any one of claims 1-17, wherein the E protein does not comprise an amino acid substitution at position 106, wherein position 106 corresponds with position 106 of SEQ ID NO: 276.
19. The stabilized flavivirus heterotetramer of any one of claims 1-18, wherein the flavivirus is selected from the group consisting of: Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus and Omsk virus.
20. The stabilized flavivirus heterotetramer of claim 19, wherein the flavivirus is Dengue virus.
21. The stabilized flavivirus heterotetramer of claim 19, wherein the flavivirus is Zika virus.
22. The stabilized flavivirus heterotetramer of claim 21, wherein the Zika virus E protein further comprises an introduced glycosylation site.
23. The stabilized flavivirus heterotetramer of claim 22, wherein the introduced glycosylation site comprises an N-X-S motif in positions 67-69, wherein the positions correspond to positions 67-69 of SEQ ID NO: 282.
24. A composition comprising: (a) a first stabilized flavivirus heterotetramer of any one of claims 1-20, wherein the flavivirus is DENV1; (b) a second stabilized flavivirus heterotetramer of any one of claims 1-20, wherein the flavivirus is DENV2; (c) a third stabilized flavivirus heterotetramer of any one of claims 1-20, wherein the flavivirus is DENV3; and (d) a fourth stabilized flavivirus heterotetramer of any one of claims 1-20, wherein the flavivirus is DENV4.
25. The composition of claim 24, further comprising: (e) a fifth stabilized flavivirus heterotetramer of any one of claims 21-23, wherein the flavivirus is ZIKV.
26. A DNA plasmid comprising a nucleic acid encoding the E protein and M protein capable of forming the stabilized flavivirus heterotetramer of any one of claims 1-23.
27. A messenger ribonucleic acid (mRNA) vaccine comprising an mRNA polynucleotide having an open reading frame (ORF) encoding a single chain protein comprising the E protein and M protein capable of forming the stabilized flavivirus heterotetramer of any one of claims 1- 23.
28. The mRNA vaccine of claim 27, comprising (a) a first mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NOs: 112, 217, 335-340, and 353-354; (b) a second mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 27, 195, 302-319, 328- 334, and 358-360; (c) a third mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 117, 175, 320-327, 341-343, and 361-363; and (d) a fourth mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 122, 229, 344-346, and 355-357.
29. The mRNA vaccine of claim 28, further comprising (e) a fifth mRNA polynucleotide comprising an ORF encoding a fusion protein comprising an amino acid sequence having at least 90% to SEQ ID NOs: 108, 140-142, 177, 285-301, and 347-352.
30. The mRNA vaccine of any one of claims 27-29, further comprising a lipid nanoparticle.
31. The mRNA vaccine of claim 30, wherein the lipid nanoparticle comprises 40-55 mol% of a lipid of Compound 1 or A7, 30-45 mol% cholesterol, 5-15 mol% 1,2 distearoyl-sn-glycero- 3-phosphocholine (DSPC), and 1-5 mol% PEG-modified lipid.
32. The mRNA vaccine of claim 30 or claim 31, wherein the lipid nanoparticle comprises Compound 1:
Figure imgf000222_0001
(Compound 1).
33. The mRNA vaccine of claim 30 or claim 31, wherein the lipid nanoparticle comprises A7:
Figure imgf000222_0002
34. The mRNA vaccine of any one of claims 31-33, wherein the PEG-modified lipid is PEG2000-DMG. 35. The mRNA vaccine of any one of claims 27-34, wherein the mRNA is fully modified with 1-methyl-pseudouridine. 36. A method of inducing an immune response in a subject, the method comprising administering to the subject one or more doses of the stabilized flavivirus heterotetramer of any one of claims 1-23, the composition of any one of claims 24-25, the plasmid DNA of claim 26, or the mRNA vaccine of any one of claims 27-35 in an effective amount to produce an immune response to a flavivirus viral infection. 37. A method of inducing an immune response in a subject, the method comprising: administering to the subject stabilized flavivirus heterotetramer of any one of claims 1-23, the composition of any one of claims 24-25, the plasmid DNA of claim 26, or the mRNA vaccine of any one of claims 27-35 in an amount effective to induce, in the subject, a population of neutralizing antibodies that cross reacts with a naturally occurring flavivirus. 38. The method of claim 36 or 37, wherein the mRNA vaccine is administered intramuscularly. 39. The method of any one of claims 36-38, wherein the flavivirus is Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, Kyasanur Forest virus, Alkhurma virus, Omsk virus, or any combination thereof. 40. The method of claim 39, wherein the flavivirus is Dengue virus. 41. The method of claim 39, wherein the flavivirus is Zika virus.
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