Engineered flavivirus antigens and uses thereof

Abstract

The present disclosure provides modified flavivirus polypeptides useful as antigens and polynucleotides encoding the same, and related compositions, methods of making, and methods of using. Also provided herein are enveloped virus-like particles and cells comprising all or a portion of said modified flavivirus polypeptides. In particular, these modified flavivirus polypeptides are useful for eliciting an immune response against flavivirus infection.

Description

Attorney Docket No.: VCCN-016 / 01WO 347597-2099ENGINEERED FLAVIVIRUS ANTIGENS AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims the priority and benefits of U.S. Provisional Application No. 63 / 687,730, filed August 27, 2024, the contents of which are incorporated by reference herein in their entirety.STATEMENT CONCERNING GOVERNMENT SUPPORT

[0002] This invention was made with government support under AY2AX000054-01 awarded by the Advanced Research Projects Agency for Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (VCCN_016_01WO_SeqList_ST26.xml; Size: 497,399 bytes; and Date of Creation: August 27, 2025) are herein incorporated by reference in their entirety.BACKGROUND

[0004] Flaviviruses are members of genus Orthofl avivirus, a group of small, enveloped, positive-strand RNA viruses. The flaviviruses contain the following three structural proteins: prM / M, the pre-membrane and membrane protein; E, the envelope protein; and C, the capsid protein. M is synthesized as a larger precursor termed prM. M and E are located in the membrane of the flavivirus particle, and so are thought to constitute important immunogenic components of the viruses. E protein has two transmembrane (TM) domains: TM1 and TM2. M protein, which closely interacts with E, similarly has two TM domains. E proteins direct flavivirus entry into cells and assembly of new virions, which bud into the ER lumen as immature, non-infectious particles with a spiky surface composed of prME heterotrimers. During egress through the trans-Golgi network of an infected cell, acid pH-induced conformational changes in E trigger cleavage of the pr portion of prM, a required step for the release of mature, infectious virus particles on which E homodimers lay flat against the viral membrane in a smooth, non-spiky conformation. The C protein complexes with the RNA to form a nucleocapsid complex. Several nonstructural proteins are also encoded by the RNA genome, including NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The genome is translatedAttorney Docket No.: VCCN-016 / 01WO 347597-2099 within the host cell as a polyprotein, then processed co- or post-translationally into the individual gene products by viral- or host-specific proteases.

[0005] There are approximately 70 members in the Orthojlavivirus genus, several of which pose current or potential threats to global public health by causing symptoms ranging from febrile illness to potentially fatal hemorrhagic or neuroinvasive disease. For example, an estimated 400 million people are infected with dengue virus (DENV) each year. Japanese encephalitis virus (JEV) is a significant public health problem, involving millions of persons at risk in the Far East. In addition, West Nile virus (WNV), which causes febrile illness, is widely distributed throughout Africa, the Middle East, the former Soviet Union, parts of Europe, and the United States.

[0006] Other flaviviruses continue to cause endemic diseases of variable nature and have the potential to emerge into new areas as a result of changes in climate, vector populations, and environmental disturbances caused by human activity. These flaviviruses include, for example, Yellow fever virus (YFV), Zika virus (ZV), Powassan virus (POWV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVEV), and Tick-borne encephalitis virus (TBEV). Other examples of flaviviruses include Spondweni virus (SPOV) and Langat virus (LGTV).

[0007] Currently, there are no approved vaccines or therapeutics that prevent broad flavivirus infection or disease. Approved vaccines and those in advanced clinical development for specific flaviviruses, such as JEV and TBEV, are primarily based on inactivated and attenuated viruses, which are generally not recommended for use during pregnancy and childbearing years. They also require longer clinical development cycles to demonstrate safety and often rely on extensively lab-adapted virus strains that are not antigenically representative of diverse circulating strains. Current approaches also fail to address the potential of cross-reactive but poorly neutralizing antibody responses, which can worsen disease outcomes. This phenomenon of antibody-dependent enhancement (ADE) of infection can be attributed to the extensive geographic overlap of multiple antigenically related flaviviruses and derailed the widespread use of the first licensed dengue vaccine. As such, there remains a need for a safe and effective genus-level flavivirus vaccine for use in broad target populations. Provided herein are compositions and methods that address this need.Attorney Docket No.: VCCN-016 / 01WO 347597-2099SUMMARY

[0008] In one aspect, disclosed herein is a modified flavivirus E polypeptide comprising at least one modification relative to a naturally occurring E polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic. In some embodiments, the at least one improved characteristic comprises increased stability, increased immunogenicity, improved breadth of immunogenicity, increased secretion as enveloped virus-like particles (eVLPs), improved ability to form a lattice, improved ability to make mosaics, improved ability to make mosaic lattices, improved expression, or decreased potential to induce antibody-dependent enhancement (ADE). In certain embodiments, the modified polypeptide exhibits increased stability in an acidic environment or at physiological temperatures.

[0009] In some embodiments, the at least one modification is ablation of an immunodominant ADE-inducing epitope within the E fusion loop and the modified polypeptide exhibits decreased potential to induce ADE.

[0010] In some embodiments, the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0011] In some embodiments, the at least one modification comprises an amino acid deletion.

[0012] In some embodiments, the at least one modification comprises an amino acid substitution.

[0013] In some embodiments, the at least one modification comprises an amino acid insertion. In some embodiments, the amino acid insertion comprises insertion of an endoplasmic reticulum (ER) signal sequence, a portion of a flavivirus nonstructural (NS) protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a potential N-linked glycosylation site (PNGS) or a combination thereof.

[0014] In some embodiments, the amino acid insertion comprises insertion of a portion of a flavivirus nonstructural protein, wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5. In certain embodiments, the flavivirus nonstructural protein is NS2 or NS3. In certain embodiments, the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD. In some embodiments, the amino acid insertion comprises insertion of an ERD. In certain embodiments, the ERD comprises the sequence of any one of SEQ ID NOS: 350-387 or a sequence having at least 70% sequence identity thereto.

[0015] In some embodiments, the insertion comprises an ER export sequence. In certain embodiments, the export sequence comprises the YTDIEM motif (SEQ ID NO: 144).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0016] In some embodiments of the modified flavivirus E polypeptide of the disclosure, the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), or Murray Valley Encephalitis virus (MVEV). In some embodiments, the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1-17 or a truncated sequence thereof.

[0017] In another aspect, provided herein is a modified NS1 polypeptide comprising at least one modification relative to a naturally occurring NS1 polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic, wherein the characteristic is increased stability, increased immunogenicity, decrease in capacity to trigger endothelial barrier permeability, improved ability to form stable oligomers for display in various forms, or increased breadth of the resultant immune response.

[0018] In some embodiments, the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0019] In some embodiments, the at least one modification comprises an amino acid deletion.

[0020] In some embodiments, the at least one modification comprises an amino acid substitution.

[0021] In some embodiments, the amino acid substitution comprises substitution of an amino acid at a position corresponding to position 115, 118, 119, or 207 of SEQ ID NO: 26.

[0022] In certain embodiments, the amino acid substitution comprises substitution of an amino acid at a position corresponding to position 115 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A).

[0023] In certain embodiments, the amino acid substitution comprises:(a) substitution of an amino acid at a position corresponding to position 118 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A);(b) substitution of an amino acid at a position corresponding to position 119 of SEQ ID NO: 26, wherein the amino acid substitution is Glycine (G) to Alanine (A);(c) substitution of an amino acid at a position corresponding to position 207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q); and / or(d) substitution of an amino acid at a position corresponding to position 207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0024] In some embodiments, the amino acid substitution comprises substitution of a wing domain of the NS1 polypeptide with a wing domain of a different flavivirus NS1 polypeptide. In certain embodiments, the wing domain of a WNV NS1 polypeptide is substituted with a wing domain of ZIKV, JEV, or DENV NS1 polypeptide. In particular embodiments, the wing domain of a different flavivirus NS1 polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 43-46, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

[0025] In some embodiments of the modified NS1 polypeptides of the disclosure, the at least one modification comprises an amino acid insertion.

[0026] In some embodiments, the amino acid insertion comprises insertion of an ER signal sequence, a portion of a flavivirus NS protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a transmembrane domain, or a combination thereof.

[0027] In some embodiments, the insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5. In certain embodiments, the flavivirus nonstructural protein is NS2 or NS3. In certain embodiments, the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD. In certain embodiments, the portion of the flavivirus nonstructural protein comprises a transmembrane domain. In particular embodiments, the transmembrane domain comprises the amino acid sequence of the transmembrane domain of any one of SEQ ID NOs: 20 to 34 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

[0028] In some embodiments, the insertion comprises an ER export sequence. In certain embodiments, the export sequence comprises the YTDIEM motif (SEQ ID NO: 144).

[0029] In some embodiments, the insertion comprises a transmembrane domain. In some embodiments, the transmembrane domain is 20, 21, 22, 23, or 24 amino acids in length. In certain embodiments, the transmembrane domain is 21 or 22 amino acids in length. In some embodiments, the transmembrane domain is derived from a bacterial or viral protein. In certain embodiments, the bacterial or viral protein is selected from the group consisting of: Epstein-Barr Virus (EBV) gp220 protein, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 M protein, Bovine viral diarrhea virus (BVDV) E2 protein, SARS-CoV-2 E protein, E. coli PmrR protein, Salmonella enterica (S. enterica) MgtU protein, Mycobacterium KdpF protein, Herpes Simplex Virus-2 (HSV-2) gH protein, Human Cytomegalovirus (HCMV) gH protein,Attorney Docket No.: VCCN-016 / 01WO 347597-2099Influenza A (FluA) M2 protein, FluA hemagglutinin protein, Measles virus hemagglutinin protein, Sendai virus (SeV) F protein, Vesicular Stomatitis Virus (VSV) G protein, Simian Vacuolating Virus 40 (SV40) Agnoprotein, Monkeypox virus (Mpox) A35R protein, and Sindbis virus (SINV) El protein.

[0030] In some embodiments, the transmembrane domain is derived from a platelet-derived growth factor receptor (PDGFR). In some embodiments, the transmembrane domain is derived from a human PDGFR (hPDGFR) or common sole PDGFR (caPDGFR). In certain embodiments, the transmembrane domain comprises the amino acid sequence of any one of SEQ ID NOS: 110-134, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

[0031] In some embodiments, the transmembrane domain is inserted at the N- or C-terminus of the NS1 polypeptide.

[0032] In some embodiments of the modified NS1 polypeptides of the disclosure comprising an amino acid insertion, the insertion comprises (a) a transmembrane (TM) domain and (b) an ERD or an ESCRT -independent eVLP inducing domain. In certain embodiments, the insertions are made at the C-terminus of the NS1 polypeptide and comprise, from N-terminus to C-terminus: (a) a TM domain and an ERD; or (b) a TM domain and an ESCRT -independent eVLP inducing domain. In some embodiments, the insertion comprises (a) a TM domain, (b) an endocytosis prevention motif (EPM), and (c) an ERD or an ESCRT -independent eVLP inducing domain. In certain embodiments, the insertions are made at the C-terminus of the NS1 polypeptide and comprise, from N-terminus to C-terminus: (a) a transmembrane (TM) domain, an EPM, and an ERD; or (b) a transmembrane (TM) domain, an EPM, and an ESCRT -independent eVLP inducing domain.

[0033] In some embodiments of the modified NS1 polypeptides of the disclosure comprising an amino acid insertion comprising an ERD, the ERD comprises the sequence of any one of SEQ ID NOS: 350-387 or a sequence having at least 70% sequence identity thereto.

[0034] In some embodiments of the modified NS1 polypeptides of the disclosure, the at least one modification is relative to a naturally occurring NS1 protein sequence from a flavivirus of the Orthoflavirvirus genus, optionally wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0035] In some embodiments, the amino acid sequence of the modified NS1 polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 20 to 34 or a truncated sequence thereof.

[0036] In some embodiments, the amino acid sequence of the modified NS1 polypeptide comprises the sequence of any one of SEQ ID NOS: 35 to 42, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto. In some embodiments, the amino acid sequence of the modified polypeptide comprises the sequence of any one of SEQ ID NOS: 278-311 and 324-336, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

[0037] In another aspect, provided herein is a plurality of modified flavivirus NS1 polypeptides selected from the modified flavivirus NS1 polypeptides of the disclosure, wherein the plurality of NS1 polypeptides are fused into a single polypeptide chain, and wherein the plurality of NS1 polypeptides comprises at least two modified NS 1 polypeptides with identical or non-identical sequences. In some embodiments, the at least two modified NS1 polypeptides comprise a NS1 polypeptide derived from a first flavivirus and a NS1 polypeptide derived from a second flavivirus. In certain embodiments, the first flavivirus and the second flavivirus are the same. In certain embodiments, the first flavivirus and the second flavivirus are different.

[0038] In some embodiments, the first flavivirus and the second flavivirus are selected independently from the group consisting of: West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0039] In some embodiments: (a) the first flavivirus is WNV and the second flavivirus is ZIKV;

[0040] (b) the first flavivirus is WNV and the second flavivirus is JEV;

[0041] (c) the first flavivirus is WNV and the second flavivirus is DENV;

[0042] (d) the first flavivirus is ZIKV and the second flavivirus is JEV;

[0043] (e) the first flavivirus is ZIKV and the second flavivirus is DENV; or

[0044] (f) the first flavivirus is JEV and the second flavivirus is DENV.

[0045] In some embodiments, a plurality of modified flavivirus NS1 polypeptides of the disclosure comprises at least three modified NS1 polypeptides with non-identical sequences, wherein the at least three NS1 polypeptides comprise a NS1 polypeptide derived from a firstAttorney Docket No.: VCCN-016 / 01WO 347597-2099 flavivirus, a NSl polypeptide derived from a second flavivirus, and a NSl polypeptide derived from a third flavivirus. In some embodiments, the first flavivirus, the second, and the third flavivirus are selected independently from the group consisting of: West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0046] In some embodiments:(a) the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is JEV;(b) the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is DENV;(c) the first flavivirus is WNV, the second flavivirus is JEV, and the third flavivirus is DENV; or(d) the first flavivirus is ZIKV, the second flavivirus is JEV, and the third flavivirus is DENV.

[0047] In some embodiments, the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides, wherein the sequences of the at least two modified NS1 polypeptides are not identical, thereby improving the breadth of immunogenicity.

[0048] In some embodiments, the plurality of modified flavivirus NS1 polypeptides of the disclosure comprises the amino acid sequence of any one of SEQ ID NOS: 286-295, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

[0049] In some embodiments, the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice.

[0050] In some embodiments, the plurality of modified flavivirus NS1 polypeptides comprises two, three, or five modified NS1 polypeptides of the disclosure, wherein the sequences of the two, three, or five modified NS1 polypeptides are not identical, optionally wherein the modified NS1 polypeptides have orthogonal interfaces.

[0051] In some embodiments, the plurality of polypeptides is capable of assembling into a stochastic mosaic, optionally comprising six, seven, or eight modified NS1 polypeptides with identical interfaces, wherein the sequences of the six, seven, or eight modified NS1 polypeptides are not identical.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0052] In another aspect, provided herein is a modified flavivirus prM polypeptide comprising at least one modification relative to a naturally occurring prM polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

[0053] In some embodiments, the characteristic is increased ability to stabilize the conformation of naturally occurring or modified flavivirus E protein, improved expression, improved secretion as eVLPs, a decrease in immature, conformationally heterogeneous virions, or a combination thereof.

[0054] In some embodiments, the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0055] In some embodiments, the at least one modification comprises an amino acid deletion. In certain embodiments, the amino acid deletion comprises deletion of the pr amino acid sequence or a portion thereof, optionally wherein the pr amino acid sequence comprises the sequence of any one of SEQ ID NOs: 70-77 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

[0056] In some embodiments, the at least one modification comprises an amino acid substitution.

[0057] In some embodiments, the at least one modification comprises an amino acid insertion.

[0058] In some embodiments, the amino acid sequence of the modified prM polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 50-67 or a truncated sequence thereof.

[0059] In some embodiments, a modified flavivirus E polypeptide of the disclosure is further fused directly or through a linker to a second flavivirus polypeptide.

[0060] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM polypeptide or a modified flavivirus prM polypeptide of the disclosure, a naturally occurring NS1 or a modified flavivirus NS1 polypeptide of the disclosure, and / or a plurality of NS1 polypeptides of the disclosure.

[0061] In some embodiments, the second flavivirus polypeptide is fused directly or through a linker to the N- or C-terminus of the modified flavivirus E polypeptide.

[0062] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM or a modified flavivirus prM polypeptide of the disclosure; wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0063] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring NS1 or a modified flavivirus NS1 polypeptide of the disclosure or the plurality of NS1 polypeptides of the disclosure; wherein the second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

[0064] In some embodiments, a modified flavivirus E polypeptide of the disclosure is further fused directly or through a linker a third flavivirus polypeptide. In certain embodiments, the third flavivirus polypeptide comprises a naturally occurring prM polypeptide or a modified flavivirus prM polypeptide of the disclosure, a modified flavivirus NS1 polypeptide of the disclosure, and / or a plurality of NS1 polypeptides of the disclosure.

[0065] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM or a modified flavivirus prM polypeptide of the disclosure and the third flavivirus polypeptide comprises a naturally occurring NS1 or a modified flavivirus NS1 polypeptide of the disclosure or a plurality of NS1 polypeptides of the disclosure; wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide and the third second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

[0066] In some embodiments, a modified flavivirus E polypeptide of the disclosure comprises a protease cleavage site. In some embodiments, the protease cleavage site is located between a modified flavivirus E polypeptide and a modified flavivirus NS1 polypeptide or a plurality of NS1 polypeptides. In some embodiments, the protease cleavage site is a naturally occurring cleavage site in a flavivirus, optionally wherein the cleavage site is a host cleavage site.

[0067] In some embodiments, a modified flavivirus E polypeptide of the disclosure comprises the sequence of any one of SEQ ID NOS: 150-173, 176-183, 185-227, 229-270, 272-277, and 312-323 or a sequence having at least 70% sequence identity thereto.

[0068] In yet another aspect, provided herein is a plurality of modified flavivirus E polypeptides of the disclosure, wherein the plurality of polypeptides are fused into a single chimeric polypeptide chain. In some embodiments, the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice. In some embodiments, the plurality of modified flavivirus E polypeptides comprises two, three, or five modified E polypeptides of the disclosure, wherein the sequences of the comprising two, three, or five modified E polypeptides are not identical, optionally wherein the modified E polypeptides have orthogonal interfaces.

[0069] In some embodiments, the plurality of polypeptides is capable of assembling into a stochastic mosaic, optionally comprising six, seven, or eight modified E polypeptides withAttorney Docket No.: VCCN-016 / 01WO 347597-2099 identical interfaces, wherein the sequences of the six, seven, or eight modified E polypeptides are not identical.

[0070] In another aspect, provided herein is a polynucleotide encoding the amino acid sequence of a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptide of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure. In some embodiments, the polynucleotide comprises DNA. In some embodiments, the polynucleotide comprises RNA. In some embodiments, the polynucleotide comprises mRNA. In certain embodiments, the mRNA comprises modified nucleosides.

[0071] In another aspect, provided herein is a vector comprising a polynucleotide of the disclosure or a plurality of polynucleotides of the disclosure.

[0072] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, adenovirus vector, modified vaccinia virus Ankara (MV A) vector, poxvirus vector, or Vesicular stomatitis virus (VSV) vector. In certain embodiments, the adenovirus vector is Ad26, Ad34, Ad35, Ad48, or Ad5HVR48.

[0073] In some embodiments, the vector is a non-viral vector. In certain embodiments, the non- viral vector is a plasmid. In certain embodiments, the non-viral vector is a lipid nanoparticle (LNP).

[0074] In another aspect, provided herein is a cell comprising a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure.

[0075] In another aspect, provided herein is a cell displaying on its cell surface all or a portion thereof of a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure.

[0076] In another aspect, provided herein is an enveloped virus-like particle (eVLP) comprising a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0077] In another aspect, provided herein is an enveloped virus-like particle (eVLP) displaying all or a portion thereof of a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure.

[0078] In another aspect, provided herein is a vaccine composition comprising a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, or an eVLP of the disclosure. In some embodiments, a vaccine composition of the disclosure further comprises one or more adjuvants. In some embodiments, a vaccine composition of the disclosure further comprising one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0079] In yet another aspect, provided herein is a kit comprising a modified flavivirus E polypeptide of the disclosure, a modified NS 1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vaccine composition of the disclosure; and instructions for use.

[0080] In yet another aspect, provided herein is a method of preventing a flavivirus infection or a symptom thereof in a subject having, or at risk of having, a flavivirus infection, comprising administering to the subject an effective amount of a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vaccine composition of the disclosure.

[0081] In some embodiments of the methods of the disclosure, the subject has, or is at risk of having, an infection of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0082] In some embodiments, the subject is a mammal or a bird, optionally wherein the subject is pregnant. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, optionally wherein the human subject is pregnant. In some embodiments, the subject is a bird.

[0083] In yet another aspect, provided herein is a method of treating a flavivirus infection or a symptom thereof in a subject having a flavivirus infection, comprising administering to the subject an effective amount of a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vaccine composition of the disclosure.

[0084] In yet another aspect, provided herein is a method of inducing anti-flavivirus antibodies in a subject, comprising administering to the subject an effective amount of a modified flavivirus E polypeptide of the disclosure, a modified NS 1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vaccine composition of the disclosure. In some embodiments, the anti- flavivirus antibodies target E protein, M protein, and / or NS1 protein, optionally wherein the anti-flavivirus antibodies target proteins of at least two flaviviruses.

[0085] In yet another aspect, provided herein is a method for inducing a flavivirus-specific immune response, comprising administering to the subject an effective amount of a modified flavivirus E polypeptide of the disclosure, a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vaccine composition of the disclosure, optionally wherein the immune response provides immune protection for at least two flaviviruses.

[0086] In yet another aspect, provided herein is a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP ofAttorney Docket No.: VCCN-016 / 01WO 347597-2099 the disclosure, or a vaccine composition of the disclosure for use in the treatment of a flavivirus infection or a symptom thereof.

[0087] Also provided herein is an use of a modified NS1 polypeptide of the disclosure, a plurality of modified flavivirus NS1 polypeptides of the disclosure, a modified prM polypeptide of the disclosure, or a plurality of modified flavivirus E polypeptides of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a cell of the disclosure, an eVLP of the disclosure, or a vaccine composition of the disclosure in the manufacture of a medicament for the treatment of a flavivirus infection or a symptom thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0088] FIG. 1 provides a schematic depiction of the generation of prME-based enveloped viruslike particles (eVLPs) .

[0089] FIG. 2 provides a schematic depiction of the generation of pr-free eVLPs, where the pr portion of prM is intentionally excluded.

[0090] FIG. 3 provides a schematic depiction of exemplary constructs of the disclosure containing engineered E protein.

[0091] FIG. 4 provides a schematic depiction of different constructs containing engineered NS1 protein and oligomers displaying epitopes that induce protective cross-flavivirus-reactive antibodies while eliminating regions thought to be involved in initiating pathogenesis.

[0092] FIG. 5 provides a schematic depiction of a process for optimizing ERDs for mRNA- delivered eVLP.

[0093] FIGS. 6A-6D provide a schematic depiction of four types of antigen designs (E-based eVLPs are shown as exemplary): eVLPs displaying E proteins as a chimeric antigen (FIG. 6A), a controlled mosaic (FIG. 6B), a stochastic mosaic (FIG. 6C), or fully orthogonal mixtures (FIG. 6D) capable of being delivered in a single mRNA vaccine formulation.

[0094] FIG. 7 provides a schematic depiction of an exemplary vaccination schedule in mice and rabbits, followed by potential immune assays used to evaluate the antisera, such as neutralization and binding assays.

[0095] FIG. 8 provides a schematic depiction for non-human primate (NHP) challenge (top) and durability (bottom) experiments.

[0096] FIGS. 9A-9B provide dot blots showing E protein expression in supernatant or in cell lysate from 293T cells transfected with plasmids encoding either prME or ME of Zika virus (ZIKV), Japanese encephalitis virus (JEV), or yellow fever virus (YFV).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0097] FIGS. 10A-10B provide dot blots showing E protein expression detected with a rabbit polyclonal anti-ZIKV E (FIG. 10A) or with a mouse monoclonal pan-fl avi virus E antibody (FIG. 10B) in supernatant (S) or from cell lysate (C) of 293T cells transfected with plasmids encoding either prME or ME of ZIKV, or YFV. Plasmids encoding prME or ME were cotransfected with those encoding ERD components (YFV NS3, ZIKV NS 1-3, or MERS-1227 spike fused to EABR).

[0098] FIGS. 11A-11B provide dot blots showing E protein expression in cell lysate (FIG. 11A) or supernatant (FIG. 11B) from 293T cells transfected with plasmids encoding components as shown in Table 8.

[0099] FIG. 12 provides a schematic of M-E-NS1 polypeptide design to enable delivery of a single construct for self-assembly of M-E and NS1 into separate eVLPs.

[0100] FIG. 13A provides a schematic of constructs representative of M-E-NS1 encoded as a single polypeptide. Asterisks indicate NS1 activity KO mutations (W115 A, W118 A, W119A, N207Q). Expi293F cells were transfected with plasmids encoding the constructs indicated in Table 9A and cell lysate and supernatant from transfected cells were analyzed by dot blot. FIGS. 13B-13C provide dot blots showing the expression and secretion of E proteins (FIG. 13B) and NS1 proteins (FIG. 13C), detected using mAb 4G2 and 22NS1, respectively, in cell lysate and supernatant from transfected cells four days post transfection.

[0101] FIG. 14A provides a schematic of ZIKV M-E-NS1 construct designs. Bars within M and E segments represent native TM domains. Expi293F cells were transfected with plasmids encoding the constructs indicated in Table 10A and cell lysate and supernatant from transfected cells were analyzed by dot blot. FIGS. 14B-14C provide dot blots showing the expression and secretion of E proteins (FIG. 14B) and NS1 proteins (FIG. 14C), detected using mAb ZV-67 and 1G65.3, respectively, in cell lysate and supernatant from transfected cells four days post transfection.

[0102] FIG. 15A provides a schematic of WNV M-E-NS1 construct designs. Expi293F cells were transfected with plasmids encoding the constructs indicated in FIG. 15A or WT WNV prME and WT WNV ME controls. Cell lysate and supernatant from transfected cells were analyzed by non-reducing SDS-PAGE and western blot. FIGS. 15B-15C provide western blots showing the expression and secretion of E proteins (FIG. 15B) and NS1 proteins (FIG. 15C), detected using mAb 4G2 and 22NS1, respectively, in cell lysate and supernatant from transfected cells four days post transfection.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0103] FIG. 16 provides a graph showing E protein expression, expressed as absorption measured at 450nm, in supernatant of Expi293F cells transfected with the indicated constructs, detected by a sandwich ELISA using anti-E mAb E16 with human Fc (hE16) for antigen capture and anti-E mAb E16 with mouse Fc (mE16) for detection four days post transfection.

[0104] FIG. 17 provides a graph showing E protein expression, expressed as absorption measured at 450nm, in supernatant of Expi293F cells transfected with the indicated constructs, detected by a sandwich ELISA using anti-E mAb EDE1-C10 with human Fc (hEDEl-ClO) for antigen capture and anti-E mAb EDE1-C10 with mouse Fc (mEDEl-ClO) for detection four days post transfection.

[0105] FIG. 18 provides a purification scheme for isolating WNV M-E-NS1 eVLPs from Expi293F suspension mammalian cells transfected with plasmids encoding a construct, for biochemical and structural characterization of the particles.

[0106] FIG. 19A provides western blot analysis of ME and NS 1 eVLPs, purified using a process as depicted in FIG. 18, under non-reducing (- BME) and reducing conditions (+ BME), eluted with and without addition of anti-E CR4354 Fab. FIG. 19B provides Coomassie stain of ME and NS1 eVLPs, purified using a process as depicted in FIG. 18, under non-reducing (- BME) and reducing (+BME) conditions. NS1 is indicated by one asterisk (*) and ME is indicated by two asterisks (**). FIG. 19C provides dynamic light scattering analysis of supernatant and pull-down samples, collected from purification process as depicted in FIG. 18.

[0107] FIG. 20 provides representative transmission electron microscopy (TEM) images of eVLPs from a M-E-NS1 design purified using a process as depicted in FIG. 18.

[0108] FIG. 21 provides a graph showing the serum dilution factor that results in 50% inhibition of infection (NT50) for each of 5 mice per group immunized with the indicated vaccine constructs. NT50 was determined from incubating serial dilutions of serum from each mouse with a standard preparation of WNV expressing a GFP reporter prior to addition to Raji cells stably expressing DC-SIGNR, a flavivirus attachment factor. Infected cells were enumerated using a high-content imager (Molecular Devices ImageXpress). Each dot on the graph shows the NT50 for each of the 5 mice per group at day 21 (gray circles; left) and day 35 (black circles; right) after immunization. Lower and upper dotted lines indicate the lower limit (LL) and upper limit (UL) of detection, respectively for assays performed with day 21 and day 35 samples.

[0109] FIG. 22A provides a graph showing the neutralization of standard (Std, solid lines) and mature (Mat, dotted lines) preparations of WNV by mAb El 6 (gray), which targets a highly exposed epitope in E domain III and mAb E53 (black), which targets the cryptic E domain IIAttorney Docket No.: VCCN-016 / 01WO 347597-2099 fusion loop epitope that is preferentially exposed on incompletely mature particles. FIG. 22B provides a graph showing the neutralization of Std (filled circles) and Mat (open circles) WNV by mouse immune sera elicited by construct designs indicated on the x-axis. Each dot represents the mean NT50 value per mouse obtained from three independent experiments in which Std and Mat WNV were tested in parallel. P-value (two-tailed) was obtained from a paired t-test.

[0110] FIGS. 23A-23C provide graphs showing the neutralization of a standard preparation of WNV (FIG. 23A), ZIKV (FIG. 23B), or DENV2 (FIG. 23C). Neutralization was measured by incubating serial dilutions of pooled sera from mice immunized with the indicated constructs, at day 49 post immunization, with a standard preparation of WNV, ZIKV, or DENV2 expressing a GFP reporter prior to addition to K562 cells, which express FcgRIIa and are poorly infected in the absence of IgG antibodies. Infected cells were enumerated by detection of GFP signal using flow cytometry.

[0111] FIGS. 24A-24B provide graphs showing binding of pooled sera to soluble E (sE) proteins of WNV (FIG. 24A) or ZIKV (FIG. 24B). Binding was measured, via a Luminex assay, by incubating serial dilutions of pooled sera collected at day 35 from mice immunized with the indicated antigens (WNVprME is also referred to as FLV.6009, WNV ME (long JEV ss) is also referred to as FLV.6013, and WNV ME-NSl-csPDGFR is also referred to as FLV.6004).

[0112] FIG. 25 provides a heatmap representing area under the curve (AUC) of MFI, measured via Luminex assay, of binding of sera to the indicated E and NS1 proteins using the Luminex platform. Sera was collected at day 35 from five mice immunized with each indicated construct, pooled at a 1 : 1 : 1 : 1 : 1 ratio, and then analyzed. Sera were assayed at dilutions of 1 : 30- 1 :2343750 using a 5-fold dilution series and the 1 : 150 and 1 :3750 dilutions were used for the summary area under the curve (AUC) calculations shown in the heatmap.

[0113] FIG. 26A provides a schematic ofNSl homodimer designs encoding a N-terminal purification tag (strep tag II) followed by SGSG linker and two copies of WNV NS1 that are also separated by a SGSG linker. The C-terminus of the second copy ofNSl is fused to the EBV gp220 TM domain and MPMV.HIV EDN tag. FIG. 26B provides a schematic of a mosaic design encoding 2 copies ofNSl. The wing domain in the second copy ofNSl is swapped with NS1 from a different flavivirus. FIG. 26C provides a schematic of heterodimeric designs encoding two copies ofNSl, each from a different flavivirus. FIG. 26D provides a schematic of heterotrimeric designs encoding three copies ofNSl, each from a different flavivirus. FIG. 26EAttorney Docket No.: VCCN-016 / 01WO 347597-2099 provides a schematic of homodimeric WNV NS1 constructs, each construct having a transmembrane domain from 16 different proteins.

[0114] FIG. 27 provides dot blots showing binding of cross-reactive mAbs to recombinant soluble flavivirus NS1 proteins, determined by spotting the indicated amounts of purified recombinant NS 1 protein from various flaviviruses on a nitrocellulose membrane and probing with anti-NSl mAb 1G5.3 (left) or 2B7 (right).

[0115] FIG. 28A provides a dot blot showing detection of NS1 protein in clarified transfection supernatant from HEK293T cells transfected with lOOng of each indicated construct. 1.5ul of clarified transfection supernatant collected three days post-transfection was blotted onto a nitrocellulose membrane and the presence of NS1 protein was probed with mAb 1G5.3, which cross-reacts with NS 1 proteins from different flaviviruses. FIG. 28B provides a graph showing the quantification of the intensity of each dot on the blot of FIG. 28 A using ImageJ software.

[0116] FIG. 29A provides a dot blot showing detection of NS1 protein in clarified transfection supernatant from HEK293T cells transfected with lOOng of each indicated construct. 1.5ul of clarified transfection supernatant collected three days post-transfection was blotted onto a nitrocellulose membrane and the presence of NS1 protein was probed with mAb 2B7, which cross-reacts with NS 1 proteins from different flaviviruses. FIG. 29B provides a graph showing the quantification of the intensity of each dot on the blot of FIG. 29 A using ImageJ software.

[0117] FIG. 30A provides a dot blot showing detection of WNV NS1 protein, measured using 22NS1 antibody, in the supernatant of HEK293T cells transfected with WNV NS1 constructs comprising the indicated transmembrane domains. FIGS. 30B-30C provide graphs showing NS1 expression on the cell surface (FIG. 30B) and NS1 total cell expression (FIG. 30C), as assessed by flow cytometry and cell ELISA, respectively.

[0118] FIG. 31 provides a graph showing predicted transmembrane domain (TM) length on the x-axis and ERD activity on the y-axis of the constructs of FIG. 30A.

[0119] FIG. 32 provides a schematic of a study design for mouse immunization with NS1 antigen designs and provides a table showing group (Grp) number of each group of mice, the construct names of the antigen designs each group received, and the platform (mRNA vs protein) used to deliver the antigens.

[0120] FIG. 33 provides a graph showing cell surface expression of NS1, assessed by flow cytometry, in Expi293F cells transfected with the indicated mRNA constructs and incubated with anti-NSl mAb 1G5.3. Bars indicate the geometric mean fluorescence intensity (gMFI) relative to mock-transfected cells.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0121] FIG. 34 provides a graph showing cell surface expression of NS1, assessed by flow cytometry, in Expi293F cells transfected with the indicated mRNA constructs and incubated with anti-NSl mAb 2B7. Bars indicate the geometric mean fluorescence intensity (gMFI) relative to mock-transfected cells.

[0122] FIG. 35 provides a dot blot showing NS 1 antigen expression and secretion, assessed by blotting cell lysate and supernatant from Expi293F cells transfected with the indicated mRNA constructs on a nitrocellulose membrane and probing with a mixture of mAbs 1G5.3 and 2B7.

[0123] FIG. 36 provides a heatmap representing area under the curve (AUC) of MFI, measured via Luminex assay, of binding of sera to the indicated NS1 proteins. Sera was collected at day 35 from five mice immunized with each indicated construct, pooled at a 1 : 1 : 1 : 1 : 1 ratio, and then analyzed. Sera were assayed at dilutions of 1 : 30- 1 :2343750 using a 5-fold dilution series and the 1 : 150 and 1 :3750 dilutions were used for the summary area under the curve (AUC) calculations shown in the heatmap.

[0124] FIG. 37 provides a schematic showing a competition ELISA for testing serum antibodies elicited by different NS 1 antigen designs.

[0125] FIG. 38A-38B provide graphs showing competitive binding between various anti-NSl mAbs and serum antibodies elicited in various groups of mice through immunization with the NS1 antigen designs indicated in FIG. 32 for said group.DETAILED DESCRIPTIONOverview

[0126] The present disclosure provides modified flavivirus polypeptides comprising an amino acid sequence derived from one or more proteins of a flavivirus, such as E protein, M protein, and / or a flavivirus non-structural protein. Also provided herein are methods for making such modified flavivirus polypeptides and methods for using these modified polypeptides to generate vaccines for the prevention or treatment of flavivirus infection. Additionally, polynucleotides encoding said modified flavivirus polypeptides, vectors comprising the same, and flavivirus vaccine compositions are also disclosed.

[0127] Non-limiting example members of the Orthojlavivirus genus from which the modified polypeptides of the disclosure are generated include, but are not limited to, West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV),Attorney Docket No.: VCCN-016 / 01WO 347597-2099 tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).Terms and Concepts

[0128] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the invention in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present invention, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present invention, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present invention and interpreted in the context of the present document and / or the accompanying figures.

[0129] Unless otherwise dictated by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry are those well-known and commonly used. Known methods and techniques are generally performed according to conventional methods well-known and as described in various general and more specific references, unless otherwise indicated. The nomenclatures used in connection with the laboratory procedures and techniques described in the present disclosure are those well-known and commonly used.

[0130] As used herein, the terms "a", "an", and "the" can refer to one or more unless specifically noted otherwise. The use of the term "or" is used to mean "and / or," unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." As used herein "another" can mean at least a second or more.

[0131] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more stepsAttorney Docket No.: VCCN-016 / 01WO 347597-2099 and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0132] The terms "about" and "approximately" as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. Exemplary degrees of error are within 20% (%); e.g., within 10%; or e.g., within 5% of a given value or range of values. Any reference to "about X" or "approximately X" specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.0IX, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions "about X" or "approximately X" are intended to teach and provide written support for a claim limitation of, for example, "0.98X." Alternatively, in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, within 5- fold, e.g. within 2-fold of a given value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value), Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0133] The term “polypeptide” is used to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to amino acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, or a non-naturally occurring amino acid. The term may be used interchangeably with the term “protein” and encompasses amino acid chains of any length, including full-length proteins or recombinant proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0134] The term "sequence identity" and the related terms and expressions used in the context of describing nucleic acid or amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequenceAttorney Docket No.: VCCN-016 / 01WO 347597-2099 comparison, typically one sequence acts as a reference sequence (subject sequence) to which test sequences (query sequence) are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0135] Depending on the algorithm, the calculated percent sequence identity may differ. For example, there are at least three ways in which to calculate a percent sequence identity.% Query sequence identity: = (Number of alignment identities) / (Length of Query sequence); % Subject sequence identity: = (Number of alignment identities) / (Length of Subject sequence); % Alignment sequence identity = (Number of alignment identities) / (Length of Alignment)

[0136] Accordingly, when the term “sequence identity” is used herein, it can include any of the above non-limiting methodologies provided above to calculate percent sequence identity.

[0137] The name of a virus, as used herein, is inclusive of all lineages and strains (historic and contemporary) of said virus unless otherwise indicated. For example, the term “Dengue virus” or “DENV” refers to a group of at least four genetically and antigenically related dengue viruses (including DENV1, DENV2, DENV3, and DENV4), unless otherwise specified. As another example, the term “West Nile virus” or “WNV” refers to a group of at least two genetically related lineages of West Nile viruses (including WNV1 and WNV2), unless otherwise specified. As yet another example, the term “Zika virus” or “ZIKV” refers to a group of Zika viruses inclusive of both historic and contemporary strains, unless otherwise specified.

[0138] The term “antibody-dependent enhancement” or “ADE” as used herein refers to phenomena characterized by non-neutralizing (or sub-optimally neutralizing) antibodies thatAttorney Docket No.: VCCN-016 / 01WO 347597-2099 facilitate virus entry into host cells, leading to increased infectivity in the cells. In some embodiments, ADE refers to a significant and detectable increase in viral infection in the presence of an antibody, relative to a pre-infection or pre-immunization sample or an unrelated antibody.

[0139] The term “antigenic” when used in the context of the present disclosure refers to the ability of a molecule to react with antibodies or effector cells. For example, a molecule that is antigenic (an antigen) may bind secreted antibodies or surface receptors on T or B cells. An antigen may or may not be elicit an immune response.

[0140] The term "immunogenic” when used in the context of the present disclosure refers to the ability of a molecule to elicit in a subject an immune response. Substances that can be immunogenic include, but are not limited to, polypeptides, proteins, polysaccharides, lipids, nucleic acids, and synthetic compounds. Various aspects of an immune response elicited by immunogenic compositions can be determined using standard assays, some of which are described in the present disclosure. It is expected that the engineered flavivirus polypeptides of the present disclosure will be immunogenic in a subject upon administration, whether they are administered as protein-based vaccines, or as nucleic acid-based vaccines.

[0141] The term “subunit vaccine” refers to a vaccine that is capable of presenting an antigen from a virus or viral particle to the immune system without introducing the complete virus or viral particle.

[0142] The terms “treatment” or “treating” used herein to refer to treatment or treating of a subject indicate any type of intervention or process performed on, or the administration of an agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In some embodiments, the disease is a disease caused by a flavivirus infection. In some embodiments, “treatment” or “treating” includes reducing the severity of one or more symptoms of a flavivirus infection. “Treatment” or “treating” also includes the use of any of the compositions or methods of the present disclosure for prophylactical treatment of a subject in need thereof. In some embodiments, a subject is treated prior to or after infection with a flavivirus. In some embodiments, a subject is treated both prior to and after infection with a flavivirus.Attorney Docket No.: VCCN-016 / 01WO 347597-2099Modified flavivirus polypeptides

[0143] In some embodiments, the modified flavivirus polypeptides of the present disclosure are themselves immunogenic or improve the immunogenicity of other antigens. Therefore, they are capable of eliciting in a subject an immune response to one or more flavivirus antigens, such as E protein, M protein, or NS1 protein. Examples of flaviviruses from which flavivirus polypeptides (including E, NS1, M, or prM polypeptides) are obtained and modified as disclosed herein include, but are not limited to, West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).Modified E polypeptides

[0144] In one aspect, described herein are modified flavivirus E polypeptides that have at least one modification relative to a naturally occurring flavivirus E polypeptide. The modification confers at least one improved characteristic on the modified E polypeptide compared to the naturally occurring E polypeptide. In some embodiments, the at least one modification is relative to a naturally occurring E protein sequence from a flavivirus of the Orthofl avivir us genus. In some embodiments, the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). Table 1 below provides exemplary naturally occurring flavivirus E polypeptides.Table 1: Exemplary naturally occurring full length E polypeptides of flavivirusesAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0145] In some embodiments, the at least one improved characteristic exhibited by the modified E polypeptide is one or more of increased stability of a E protein, increased immunogenicity, improved breadth of immunogenicity, increased secretion as enveloped virus-like particles (eVLPs), improved ability to form a lattice, improved ability to make mosaics, improved ability to make mosaic lattices, improved protein expression, or decreased potential to induce antibodydependent enhancement (ADE). Mosaics refer to an assembly of modified polypeptides derived from at least two flaviviruses. Mosaic designs may elicit a broader immune response. In some embodiments, the modified E polypeptide exhibits increased stability in an acidic environment or at physiological temperatures. In certain embodiments where the at least one improved characteristic is decreased potential to induce ADE, the modification comprises ablation of an immunodominant ADE-inducing epitope within the E fusion loop.

[0146] Modifications that can be made to the flavivirus E polypeptide include, but are not limited to, a mutation, a deletion, an insertion, and / or an amino acid substitution. In some embodiments, the at least one modification made to the modified flavivirus E polypeptide of the present disclosure comprises an amino acid deletion. In some embodiments, the at least one modification comprises an amino acid substitution.

[0147] In some embodiments, the at least one modification comprises an amino acid insertion. Insertion sites may include, but are not limited to, (1) between the first transmembrane helix (TM1) and the second transmembrane helix (TM2) of an E polypeptide and (2) at the truncated C terminus of an E polypeptide after deletion of TM2. In some embodiments, the insertion site isAttorney Docket No.: VCCN-016 / 01WO 347597-2099 between TM1 and TM2 of an E polypeptide. In some embodiments, TM2 of E is deleted and the sequence inserted at the truncated C terminus of E polypeptide.

[0148] Insertions may include, but are not limited to, an amino acid sequence of an endoplasmic reticulum (ER) signal sequence, a portion of a flavivirus nonstructural (NS) protein, an ER export sequence, an endosomal sorting complexes required for transport (ESCRT) recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a transmembrane domain, a potential N-linked glycosylation site (PNGS), an endocytosis prevention motif (EPM), a purification tag, or a combination thereof.

[0149] In some embodiments, the insertion comprises insertion of an ER signal sequence. An ER signal sequence may comprise the signal sequence from JEV strain SAI 4 or JEV strain India / 78. Table 6E below provides exemplary ER signal sequences, which are also referred to as signal sequences herein. In some embodiments, the signal sequence comprises the sequence of any one of SEQ ID NOS: 140-144, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto. An ER signal sequence is also referred to as a signal sequence herein.

[0150] In some embodiments, the insertion comprises insertion of an ERD. In some embodiments, the ERD comprises a sequence in Table 5A or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOs: 350-387 or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0151] In some embodiments, the insertion comprises insertion of an ESCRT-independent eVLP inducing domain. Examples of ESCRT-independent eVLP inducing domains include, but are not limited to, the platelet-derived growth factor receptor (PDGFR) transmembrane domain or amphipathic helices. In some embodiments, the ESCRT-independent eVLP inducing domain comprises the amino acid sequence of any one of SEQ ID NOs: 110-112 or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at leastAttorney Docket No.: VCCN-016 / 01WO 347597-209990%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0152] In some embodiments, the amino acid insertion comprises insertion of a portion of a flavivirus nonstructural (NS) protein. In some embodiments, the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5. In some embodiments, the NS protein is NS2 or NS3. In certain embodiments, the portion of the flavivirus NS protein that is inserted into the modified E polypeptide comprises a transmembrane domain or an ERD. In certain embodiments, the portion of the flavivirus NS protein that is inserted comprises a transmembrane domain of a flavivirus protein or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto.

[0153] In some embodiments, the amino acid insertion comprises insertion of an ER export sequence. In some embodiments, the ER export sequence comprises the YTDIEM motif (SEQ ID NO: 145). Without being held to theory or mechanism the YTDIEM motif is found in the cytoplasmic tail of the vesicular stomatitis virus G protein and functions as an ER export sequence. The YTDIEM motif can be replaced with a stretch of alanine. In some embodiments, the YTDIEM motif is replaced with 10-15 alanines. In some embodiments, the ER export sequence comprises 10-15 alanines.

[0154] In some embodiments, the insertion comprises insertion of an EPM. In some embodiments, the EPM is derived from a non-human EPM. In some embodiments, the EPM is derived from a mouse, arctic fox, or common degu EPM. In some embodiments, the EPM comprises the sequence of any one of SEQ ID NOS: 107-109 or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0155] Inclusion of a potential N-linked glycosylation site (PNGS) in pr or E has been shown to impair ER export of E protein. Without being held to theory or mechanism, N-linked glycosylation sites (PNGS) may be added to a modified flavivirus E polypeptides described herein to facilitate eVLP generation. Without being held to theory or mechanism, PNGS may recruit ERGIC53, a mannose-specific membrane lectin that acts as cargo receptor for ER to Golgi transport.

[0156] In some embodiments, a modified flavivirus E polypeptide of the disclosure comprises the amino acid sequence of a naturally occurring E polypeptide of Table 1, or a sequence havingAttorney Docket No.: VCCN-016 / 01WO 347597-2099 at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, a modified flavivirus E polypeptide of the disclosure comprises the amino acid sequence of any one of SEQ ID NOS: 1-17, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0157] In some embodiments, the modified flavivirus E polypeptide of the present disclosure is further fused to a second flavivirus polypeptide. In some embodiments, the modified flavivirus E polypeptide is fused directly to a second flavivirus polypeptide. In some embodiments, the modified flavivirus E polypeptide is fused through a linker to a second flavivirus polypeptide. In some embodiments, the modified flavivirus E polypeptide is fused through protease cleavage site to a second flavivirus polypeptide.

[0158] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM or a modified flavivirus prM polypeptide described herein, a naturally occurring NS1 or a modified NS1 polypeptide described herein, and / or a plurality of NS1 polypeptides described herein. In some embodiments, the second flavivirus polypeptide is fused directly or through a linker to the N- or C-terminus of the modified flavivirus E polypeptide. In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM or a modified flavivirus prM polypeptide described herein. In some embodiments, the second flavivirus polypeptide comprises a naturally occurring NS1 or a modified NS1 polypeptide described herein. In some embodiments, the second flavivirus polypeptide comprises a plurality of NS1 polypeptides described herein.

[0159] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM or a modified flavivirus prM polypeptide of the disclosure, wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide. In some embodiments, the second flavivirus polypeptide comprises a naturally occurring NS1 or a modified flavivirus NS1 polypeptide of the disclosure or a plurality of NS1 polypeptides of the disclosure, wherein the second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

[0160] In some embodiments, the modified flavivirus E polypeptide of the present disclosure is further fused directly or through a linker to a second and third flavivirus polypeptide. In someAttorney Docket No.: VCCN-016 / 01WO 347597-2099 embodiments, the third flavivirus polypeptide comprises a naturally occurring prM polypeptide or a modified flavivirus prM polypeptide of the disclosure, a modified flavivirus NS1 polypeptide of the disclosure, and / or a plurality of NS1 polypeptides of the disclosure.

[0161] In some embodiments, the second flavivirus polypeptide comprises a naturally occurring prM or a modified flavivirus prM polypeptide of the disclosure and the third flavivirus polypeptide comprises a naturally occurring NS1 or a modified flavivirus NS1 polypeptide of the disclosure or a plurality of NS1 polypeptides of the disclosure, wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide and the third second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

[0162] In some embodiments, the modified flavivirus E polypeptide fused to a second flavivirus polypeptide further comprises a protease cleavage site, wherein the protease cleavage site is located between the modified flavivirus E polypeptide and the second flavivirus polypeptide. In some embodiments, the modified flavivirus E polypeptide fused to a second and a third flavivirus polypeptide further comprises a protease cleavage site, wherein the protease cleavage site is located between the modified flavivirus E polypeptide and the second flavivirus polypeptide or between the modified flavivirus E polypeptide and the third flavivirus polypeptide. FIG. 12 provides a schematic depicting a construct design comprising a modified flavivirus E polypeptide comprising a protease cleavage site.

[0163] In some embodiments, the protease cleavage site is located between the modified flavivirus E polypeptide and the second flavivirus polypeptide, wherein the second flavivirus polypeptide comprises a modified flavivirus NS1 polypeptide of the disclosure or a plurality of NS1 polypeptides of the disclosure.

[0164] In some embodiments, the protease cleavage site is a naturally occurring protease cleavage site between M, E, and / or NS 1 polypeptides of a flavivirus. In some embodiments, the protease cleavage site is a naturally occurring cleavage site within the junctions between the M, E, and / or NS1 proteins. In some embodiments, the protease cleavage site is a host protease cleavage site, wherein the protease cleavage site is not cleaved by a viral (e.g., a flavivirus) protease and is cleaved by a protease from a host cell with which a modified polypeptide comes into contact. In some embodiments, the host cell is a cell of a subject described herein. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is an immortalized cell. In some embodiments, the protease from a host cell is a signal peptidase.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0165] Without being held to theory or mechanism, in some embodiments, a protease cleavage site allows for proper cleavage of individual polypeptides, thereby enabling self-assembly of ME into eVLPs and assembly of NS1 polypeptides into eVLPs driven through ESCRT-dependent or independent mechanisms.

[0166] Exemplary amino acid sequences of a modified flavivirus E polypeptide of the disclosure fused to a second polypeptide are provided in Tables 6A-6C. In some embodiments, said amino acid sequences comprise the sequence of any one of SEQ ID NOs: 150-173, 176-183, 185-227, 229-270, 272-277, and 312-323 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0167] Also disclosed herein is a plurality (two or more) of the modified flavivirus E polypeptides described herein. In some embodiments, the plurality of modified flavivirus E polypeptides described herein is fused to form a single chain chimeric polypeptide. In some embodiments, the plurality of modified E polypeptides comprises at least two modified E polypeptides with identical sequences. In some embodiments, the plurality of modified E polypeptides comprises at least two modified E polypeptides, wherein the sequences of the at least two modified E polypeptides are not identical. For example, a chimeric E polypeptide may comprise modified E polypeptides from two or more different flaviviruses. In some embodiments, the plurality of the modified flavivirus E polypeptides form dimers. In some embodiments, the plurality of the modified flavivirus E polypeptides form homodimers. In some embodiments, the plurality of the modified flavivirus E polypeptides form heterodimers. In some embodiments, the plurality of the modified flavivirus E polypeptides is capable of assembling into a lattice of anti-parallel dimers (e.g. on an eVLP surface), occupying a 5-fold, 3- fold, or 2-fold axis of symmetry.

[0168] In some embodiments, the plurality of modified flavivirus E polypeptides comprises at least two modified E polypeptides described herein, wherein the sequences of the two, three, or five modified E polypeptides are not identical. In certain embodiments, the plurality of modified flavivirus E polypeptides comprises two, three, or five modified E polypeptides described herein, wherein the sequences of the two, three, or five modified E polypeptides are not identical. When one protein interacts with another protein, the area where they contact each other is termed the protein-protein interface. Interface residues of a protein are the amino acid residues that contact with the amino acid residues from an interacting protein. In someAttorney Docket No.: VCCN-016 / 01WO 347597-2099 embodiments, the interface amino acid residues of each non-identical modified E polypeptides of the plurality of modified E polypeptides are different, thereby defining their interaction or lack thereof with other polypeptides.

[0169] As used herein, modified polypeptides with interface residues that can interact with each other are referred to as an orthogonal pair and their interface is referred to as an orthogonal interface. Each modified E polypeptide described herein may form an orthogonal pair with more than one other modified E polypeptide. In some embodiments, the plurality of modified E polypeptides described herein have orthogonal interfaces, and can organize into a controlled mosaic. In other embodiments, the plurality of modified flavivirus E polypeptides described herein do not have orthogonal interfaces, and form a stochastic mosaic where the interaction between the modified polypeptides is random. Controlled mosaic and stochastic mosaics are further described below. In some embodiments, the plurality of modified flavivirus E polypeptides comprises six, seven, or eight modified E polypeptides, wherein the sequences of the six, seven, or eight modified E polypeptides are not identical. In certain embodiments, the plurality of modified E polypeptides have identical interfaces.Modified NS1 polypeptide

[0170] In another aspect, described herein are modified NS1 polypeptides comprising at least one modification relative to a naturally occurring NS1 polypeptide. The modification confers at least one improved characteristic on the modified NS1 polypeptide compared to the naturally occurring NS1 polypeptide. In some embodiments, the at least one modification is relative to a naturally occurring NS1 protein sequence from a flavivirus of the Orthoflavivirus^enus. In some embodiments, the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). Table 2A below provides exemplary naturally occurring NS1 polypeptide of several flaviviruses.Table 2A: Exemplary naturally occurring NS1 polypeptides of flavivirusesAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0171] In some embodiments, the at least one improved characteristic exhibited by the modified NS1 polypeptide is increased stability, increased secretion as enveloped virus-like particles (eVLPs), increased immunogenicity, decrease in capacity to trigger endothelial barrier permeability, improved ability to form stable oligomers for display in various forms, or increased breadth of the resultant immune response.

[0172] Modifications that can be made to the flavivirus NS1 polypeptide include, but are not limited to, a mutation, a deletion, an insertion, and / or an amino acid substitution. In some embodiments, the at least one modification made to the modified flavivirus NS1 polypeptide of the present disclosure comprises an amino acid deletion.

[0173] In some embodiments, the at least one modification comprises an amino acid substitution. In some embodiments, the amino acid substitution is at a position corresponding to position 115, 118, 119, or 207 of SEQ ID NO: 26.

[0174] In some embodiments, the amino acid substitution is at a position corresponding to position 115 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A). In some embodiments, the amino acid substitution is at a position corresponding to position 118 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A). In some embodiments, the amino acid substitution is at a position corresponding to position 119 of SEQ ID NO: 26, wherein the amino acid substitution is Glycine (G) to Alanine (A). In some embodiments, the amino acid substitution is at a position corresponding to positionAttorney Docket No.: VCCN-016 / 01WO 347597-2099207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q). In some embodiments, the amino acid substitution is at positions corresponding to positions 115, 118, 119, and 207 of SEQ ID NO: 26, wherein the amino acid substitution is W to A, W to A, G to A, and N to Q, respectively.

[0175] In some embodiments, the amino acid substitution comprises substitution of the wing domain of a WNV NS1 polypeptide with the wing domain of a different NS1 polypeptide. In some embodiments, the amino acid substitution comprises substitution of the wing domain of a WNV NS1 polypeptide with the wing domain of ZIKV, JEV, or DENV NS 1 polypeptide. In certain embodiments, the amino acid substitution comprises substitution of the wing domain of a WNV NS1 polypeptide with a wing domain comprising the amino acid sequence of any one of SEQ ID NOS: 43-46, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0176] In some embodiments, the at least one modification comprises an amino acid insertion. Insertions may include, but are not limited to, insertion of an ER signal sequence, a portion of a flavivirus NS protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT- independent eVLP inducing domain, a transmembrane domain, an endocytosis prevention motif (EPM), a purification tag, or a combination thereof.

[0177] In some embodiments, the insertion comprises insertion of an ER signal sequence. An ER signal sequence may comprise the signal sequence from JEV strain SAI 4 or JEV strain India / 78. Table 6E below provides exemplary signal sequences. In some embodiments, the signal sequence comprises the sequence of any one of SEQ ID NOS: 140-144, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto. An ER signal sequence is also referred to as a signal sequence herein.

[0178] In some embodiments, the insertion comprises insertion of an ERD. In some embodiments, the ERD comprises a sequence in Table 5A or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ERD comprises the amino acid sequence of any one of SEQ ID NOs: 350-387 or a sequence having at least 1, 2, 3,Attorney Docket No.: VCCN-016 / 01WO 347597-20994, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0179] In some embodiments, the insertion comprises insertion of an ESCRT-independent eVLP inducing domain. Examples of ESCRT-independent eVLP inducing domains include, but are not limited to, the platelet-derived growth factor receptor (PDGFR) transmembrane domain or amphipathic helices. In some embodiments, the ESCRT-independent eVLP inducing domain comprises the amino acid sequence of any one of SEQ ID NOs: 110-112 or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0180] In some embodiments, the insertion comprises insertion of an EPM. In some embodiments, the EPM is derived from a non-human EPM. In some embodiments, the EPM is derived from a mouse, arctic fox, or common degu EPM. In some embodiments, the EPM comprises the sequence of any one of SEQ ID NOS: 107-109 or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0181] In some embodiments, the amino acid insertion comprises insertion of a portion of a flavivirus nonstructural (NS) protein. In some embodiments, the flavivirus NS protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5. In some embodiments, the NS protein is NS2 or NS3. In certain embodiments, the portion of the flavivirus NS protein that is inserted into the modified NS1 polypeptide comprises a transmembrane domain or an ERD. In certain embodiments, the portion of the flavivirus NS protein that is inserted comprises a transmembrane domain comprising the amino acid sequence of the TM sequence of any one of SEQ ID NOs: 16 to 30 or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0182] In some embodiments, the amino acid insertion comprises insertion of an ER export sequence. In some embodiments, the ER export sequence comprises the YTDIEM motif (SEQ ID NO: 145). Without being held to theory or mechanism the YTDIEM motif is found in the cytoplasmic tail of the vesicular stomatitis virus G protein and functions as an ER export sequence. The YTDIEM motif can be replaced with a stretch of alanine. In some embodiments, the YTDIEM motif is replaced with 10-15 alanines. In some embodiments, the ER export sequence comprises 10-15 alanines.

[0183] In some embodiments, the amino acid insertion comprises insertion of a transmembrane domain. In some embodiments, the transmembrane domain is 20, 21, 22, 23, or 24 amino acids in length. In certain embodiments, the transmembrane domain is 21 or 22 amino acids in length.

[0184] In some embodiments, the transmembrane domain is derived from a bacterial or viral protein. In certain embodiments, the bacterial or viral protein is selected from the group consisting of: Epstein-Barr Virus (EBV) gp220 protein, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 M protein, Bovine viral diarrhea virus (BVDV) E2 protein, SARS- CoV-2 E protein, E. coli PmrR protein, Salmonella enterica (S. enterica) MgtU protein, Mycobacterium KdpF protein, Herpes Simplex Virus-2 (HSV-2) gH protein, Human Cytomegalovirus (HCMV) gH protein, Influenza A (FluA) M2 protein, FluA hemagglutinin protein, Measles virus hemagglutinin protein, Sendai virus (SeV) F protein, Vesicular Stomatitis Virus (VSV) G protein, Simian Vacuolating Virus 40 (SV40) Agnoprotein, Monkeypox virus (Mpox) A35R protein, and Sindbis virus (SINV) El protein.

[0185] In some embodiments, the transmembrane domain is derived from a platelet-derived growth factor receptor (PDGFR). In certain embodiments, the transmembrane domain is derived from a human PDGFR (hPDGFR) or common sole PDGFR (caPDGFR).

[0186] In some embodiments, the transmembrane domain comprises an amino acid sequence in Table 6D, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the transmembrane domain is inserted at the N- or C-terminus of the NS1 polypeptide.

[0187] In some embodiments, the transmembrane domain comprises the amino acid sequence of any one of SEQ ID NOS: 110-134, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, atAttorney Docket No.: VCCN-016 / 01WO 347597-2099 least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the transmembrane domain is inserted at the N- or C-terminus of the NS1 polypeptide.

[0188] In some embodiments, the amino acid insertion comprises insertion of (a) a transmembrane (TM) domain and (b) an ERD or an ESCRT -independent eVLP inducing domain. In some embodiments, the insertions are made at the N- or C-terminus of the NS1 polypeptide. In some embodiments, the insertions are made at the N-terminus of the NS1 polypeptide. In some embodiments, the insertions are made at the C-terminus of the NS1 polypeptide. In some embodiments, the insertions comprise, from N-terminus to C-terminus, (a) a TM domain and an ERD; or (b) a TM domain and an ESCRT -independent eVLP inducing domain.

[0189] In some embodiments, the amino acid insertion comprises insertion of (a) a TM domain, (b) an endocytosis prevention motif (EPM), and (c) an ERD or an ESCRT -independent eVLP inducing domain. In some embodiments, the insertions are made at the N- or C-terminus of the NS1 polypeptide. In some embodiments, the insertions are made at the N terminus of the NS 1 polypeptide. In some embodiments, the insertions are made at the C-terminus of the NS1 polypeptide. In some embodiments, the insertions comprise, from N-terminus to C-terminus, (a) a transmembrane (TM) domain, an EPM, and an ERD; or (b) a transmembrane (TM) domain, an EPM, and an ESCRT -independent eVLP inducing domain.

[0190] In some embodiments, a modified flavivirus NS1 polypeptide of the disclosure comprises the amino acid sequence of a naturally occurring NS1 polypeptide of Table 2A, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, a modified flavivirus NS1 polypeptide of the disclosure comprises the amino acid sequence of any one of SEQ ID NOS: 20-34, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0191] Table 2B below provides amino acid sequences for exemplary modified flavivirus NS1 polypeptides described herein.Table 2B: Exemplary modified flavivirus NS1 polypeptidesAttorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0192] In some embodiments, a modified flavivirus NS1 polypeptide of the disclosure comprises an amino acid sequence in Table 2B, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, a modified flavivirus NS1 polypeptide of the disclosure comprises the amino acid sequence of any one of SEQ ID NOS: 35-42, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0193] Also disclosed herein is a plurality of the modified flavivirus NS1 polypeptides described herein. In some embodiments, the plurality of modified flavivirus NS1 polypeptides described herein is fused to form a single chain chimeric polypeptide. For example, a chimeric NS1 polypeptide may comprise modified NS1 polypeptides of two or more flaviviruses.

[0194] In some embodiments, the plurality of modified NS 1 polypeptides comprises at least two modified NS 1 polypeptides with identical sequences. In some embodiments, the plurality of modified NS1 polypeptides comprises at least two modified NS1 polypeptides wherein the sequences of the at least two modified NS1 polypeptides are not identical. In some embodiments, the at least two modified NS1 polypeptides comprise a NS1 polypeptide derived from a first flavivirus and a NS1 polypeptide derived from a second flavivirus. In certain embodiments, the first flavivirus and the second flavivirus are the same. In certain embodiments, the first flavivirus and the second flavivirus are different. In some embodiments, the first flavivirus and the second flavivirus are selected independently from the group consisting of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitisAttorney Docket No.: VCCN-016 / 01WO 347597-2099 virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). In certain embodiments, the first flavivirus is WNV and the second flavivirus is ZIKV. In certain embodiments, the first flavivirus is WNV and the second flavivirus is JEV. In certain embodiments, the first flavivirus is WNV and the second flavivirus is DENV. In certain embodiments, the first flavivirus is ZIKV and the second flavivirus is JEV. In certain embodiments, the first flavivirus is ZIKV and the second flavivirus is DENV. In certain embodiments, the first flavivirus is JEV and the second flavivirus is DENV.

[0195] In some embodiments, the plurality of modified NS 1 polypeptides comprises at least three modified NS1 polypeptides with identical sequences. In some embodiments, the plurality of modified NS1 polypeptides comprises at least three modified NS1 polypeptides with nonidentical sequences, wherein the at least three NS1 polypeptides comprise a NS1 polypeptide derived from a first flavivirus, a NS1 polypeptide derived from a second flavivirus, and a NS1 polypeptide derived from a third flavivirus. In some embodiments, the first, the second, and the third flaviviruses are selected independently from the group consisting of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). In certain embodiments, the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is JEV. In certain embodiments, the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is DENV. In certain embodiments, the first flavivirus is WNV, the second flavivirus is JEV, and the third flavivirus is DENV. In certain embodiments, the first flavivirus is ZIKV, the second flavivirus is JEV, and the third flavivirus is DENV.

[0196] In some embodiments, the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides with different sequences, thereby improving the breadth of immunogenicity.

[0197] In some embodiments, the plurality of NS1 polypeptides comprises the amino acid sequence of any one of SEQ ID NOS: 286-295, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0198] In some embodiments, the plurality of the modified flavivirus NS1 polypeptides form dimers. In some embodiments, the plurality of the modified flavivirus NS1 polypeptides form homodimers. In some embodiments, the plurality of the modified flavivirus NS1 polypeptides form heterodimers. In some embodiments, the plurality of the modified flavivirus NS1 polypeptides is capable of assembling into a lattice of anti-parallel dimers (e.g. on an eVLP surface), occupying a 5-fold, 3-fold, or 2-fold axis of symmetry.

[0199] In some embodiments, the plurality of modified flavivirus NS1 polypeptides comprises at least two modified NS1 polypeptides described herein, wherein the sequences of the at least two modified NS1 polypeptides are not identical. In certain embodiments, the plurality of modified flavivirus NS1 polypeptides comprises two, three, or five modified NS1 polypeptides described herein, wherein the sequences of the two, three, or five modified NS1 polypeptides are not identical. When one protein interacts with another protein, the area where they contact each other is termed the protein-protein interface. Interface residues of a protein are the amino acid residues that contact with the amino acid residues from an interacting protein. In some embodiments, the interface amino acid residues of each non-identical modified NS1 polypeptides of the plurality of modified NS1 polypeptides are different, thereby defining their interaction or lack thereof with other polypeptides.

[0200] Modified NS1 polypeptides with interface residues that can interact with each other are referred to as an orthogonal pair and their interface is referred to as an orthogonal interface. Each modified NS1 polypeptide described herein may form an orthogonal pair with more than one other polypeptide. In some embodiments, the plurality of modified NS1 polypeptides described herein have orthogonal interfaces, and can organize into a controlled mosaic. In other embodiments, the plurality of modified flavivirus NS1 polypeptides described herein do not have orthogonal interfaces, and form a stochastic mosaic where the interaction between the modified polypeptides is random. Controlled mosaic and stochastic mosaics are further described below. In some embodiments, the plurality of modified flavivirus NS1 polypeptides comprises six, seven, or eight modified NS1 polypeptides, wherein the sequences of the six, seven, or eight modified NS1 polypeptides are not identical. In certain embodiments, the plurality of modified NS1 polypeptides have identical interfaces.

[0201] In some embodiments, the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice. In some embodiments, the plurality of polypeptides comprises two, three, or five modified NS1 polypeptides of the disclosure, wherein the sequences of the two,Attorney Docket No.: VCCN-016 / 01WO 347597-2099 three, or five modified NS1 polypeptides are not identical. In certain embodiments, the modified NS1 polypeptides have orthogonal interfaces.

[0202] In some embodiments, the plurality of polypeptides is capable of assembling into a stochastic mosaic. In certain embodiments, the plurality of polypeptides comprises six, seven, or eight modified NS 1 polypeptides with identical interfaces wherein the sequences of the six, seven, or eight modified NS1 polypeptides are not identical.Modified prM polypeptides

[0203] In another aspect, described herein are modified flavivirus precursor prM polypeptides that have at least one modification relative to a naturally occurring flavivirus prM polypeptide. The modification confers at least one improved characteristic on the modified prM polypeptide compared to the naturally occurring prM polypeptide. In some embodiments, the at least one modification is relative to a naturally occurring prM protein sequence from a flavivirus of the Orthoflavivirus genus. In some embodiments, the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickbome encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). A naturally occurring prM polypeptide, as used herein, refers to a polypeptide comprising a naturally occurring pr polypeptide and a naturally occurring M polypeptide. For example, a naturally occurring prM polypeptide of ZIKV comprises the ZIKV M polypeptide set forth in SEQ ID NO: 50 and the ZIKV pr polypeptide set forth in SEQ ID NO: 75. The term modified flavivirus prM polypeptide as used herein is inclusive of modified prM polypeptides wherein the amino acid sequence of pr is deleted in part or in its entirety. The term pr polypeptide as used herein refers to a pr polypeptide which does not contain an M polypeptide or any portion thereof. The term M polypeptide as used herein refers to a M polypeptide which does not contain a pr polypeptide or any portion thereof.

[0204] Table 3A below provides exemplary naturally occurring M polypeptides of several flaviviruses.Table 3A: Exemplary naturally occurring M polypeptides of flavivirusesAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0205] Table 3B below provides exemplary naturally occurring pr polypeptides of several flaviviruses.Table 3B: Exemplary naturally occurring pr polypeptide of flaviviruses

[0206] In some embodiments, the at least one improved characteristic exhibited by the modified prM polypeptide is increased ability to stabilize the conformation of naturally occurring or modified flavivirus prM or M protein, improved expression of M protein, increased secretion asAttorney Docket No.: VCCN-016 / 01WO 347597-2099 enveloped virus-like particles (eVLPs), a decrease in immature, conformationally heterogeneous virions, or a combination thereof.

[0207] Modifications that can be made to the flavivirus prM polypeptide include, but are not limited to, a mutation, a deletion, an insertion, and / or an amino acid substitution. In some embodiments, the at least one modification comprises an amino acid substitution. In some embodiments, the at least one modification comprises an amino acid insertion.

[0208] As described in the background section above, the pr portion of prM is removed when prM is processed to form M protein in mature virions. Cleavage of pr can be inefficient such that secreted virions are structurally heterogeneous, composed of immature, partially mature, and fully mature forms. Heterogeneity in flavivirus maturation state may impact E protein antigenicity. Without being held to theory or mechanism, the presence of uncleaved pr in incompletely mature virions may expose E protein epitopes such as the fusion loop, which is often targeted by antibodies that enhance rather than neutralize flavivirus infection.

[0209] Additionally, antibodies targeting uncleaved pr itself may mediate antibody-dependent enhancement (ADE) and contribute to disease severity. Moreover, neutralizing antibody responses against mature virions have been shown to provide protective immunity following vaccination and natural infection.

[0210] Therefore, and without being held to theory or mechanism, in some embodiments it is advantageous to delete the pr sequence from the modified prM polypeptide of the present disclosure to reduce the inefficiencies associated with pr cleavage and to enable generation of mature viral particles.

[0211] In some embodiments, the at least one modification made to the modified flavivirus prM polypeptide of the present disclosure comprises an amino acid deletion. In some embodiments, the amino acid deletion includes deletion of the amino acid sequence of pr, or a portion thereof. In certain embodiments, the amino acid sequence of pr comprises a sequence in Table 3B, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In specific embodiments, the amino acid sequence of pr comprises any one of SEQ ID NOS: 70-77, or a sequence having at least 1, 2, 3, 4, or 5 amino acid variations thereto, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, atAttorney Docket No.: VCCN-016 / 01WO 347597-2099 least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0212] In some embodiments, the at least one modification made to the modified flavivirus prM polypeptide of the present disclosure comprises an amino acid insertion. Insertions may include, but are not limited to, insertion of an ER signal sequence, a portion of a flavivirus NS protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a transmembrane domain, an endocytosis prevention motif (EPM), a purification tag, or a combination thereof.

[0213] In some embodiments, a modified flavivirus prM polypeptide of the disclosure comprises the amino acid sequence of a naturally occurring M or pr polypeptide of Table 3A or Table 3B, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the amino acid sequence of the modified prM polypeptide disclosed herein comprises the sequence of any one of SEQ ID NOs: 50-66 and 70-88, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0214] In some embodiments, a modified flavivirus prM polypeptide of the disclosure comprises the amino acid sequence of a naturally occurring M polypeptide of Table 3A, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the amino acid sequence of the modified prM polypeptide disclosed herein comprises the sequence of any one of SEQ ID NOs: 50-66, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0215] Table 3A provides exemplary naturally occurring M polypeptide of several flaviviruses.Exemplary Configurations

[0216] In some embodiments, a modified flavivirus polypeptide of the disclosure comprises:(a) a signal sequence;(b) a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein;Attorney Docket No.: VCCN-016 / 01WO 347597-2099(c) a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein;(d) a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein;(e) a transmembrane domain described herein;(f) a endocytosis prevention motif (EPM); and / or(g) an ERD or an ESCRT -independent eVLP inducing domain.

[0217] In some embodiments, the order in which (a) - (g), if present, are fused together is according to the depictions provided in FIG. 12, FIG. 13A, FIG. 14A, FIG. 15A, or FIGS.26A-26E

[0218] In some embodiments, a modified flavivirus polypeptide of the disclosure comprises two or more copies of a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein. In some embodiments, a modified flavivirus polypeptide of the disclosure comprises two or more copies of any one of (a) - (g). In some embodiments, (a) - (g), if present, are fused together directly, through a linker, or through a protease cleavage site.

[0219] In some embodiments, a modified flavivirus polypeptide of the disclosure further comprises a purification tag. In some embodiments, the purification tag binds to streptavidin or engineered streptavidin with high affinity. In certain embodiments, the engineered streptavidin is Strep-Tactin. In some embodiments, the purification tag is Strep-tag II. A purification tag, when included in the modified flavivirus polypeptides of the disclosure, can be used for detection and / or purification of said polypeptides.

[0220] In some embodiments, a modified flavivirus polypeptide of the disclosure comprises, from N- to C- terminus:(a) a signal sequence, a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein, a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a transmembrane domain described herein, a endocytosis prevention motif (EPM) described herein, and a ERD or an ESCRT -independent eVLP inducing domain;(b) a signal sequence, a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein, a protease cleavage site, a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein,Attorney Docket No.: VCCN-016 / 01WO 347597-2099 a transmembrane domain described herein, a endocytosis prevention motif (EPM) described herein, and a ERD or an ESCRT -independent eVLP inducing domain;(c) a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein, a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a transmembrane domain described herein, a endocytosis prevention motif (EPM) described herein, and a ERD or an ESCRT - independent eVLP inducing domain;(d) a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein, a protease cleavage site, a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a transmembrane domain described herein, a endocytosis prevention motif (EPM) described herein, and a ERD or an ESCRT -independent eVLP inducing domain;(e) a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(f) a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a ERD or an ESCRT -independent eVLP inducing domain, and a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein;(g) a signal sequence, a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein, and an ERD or an ESCRT-independent eVLP inducing domain;(h) a signal sequence, a naturally occurring flavivirus prM polypeptide or a modified flavivirus prM polypeptide described herein, a ERD or an ESCRT-independent eVLP inducing domain, and a naturally occurring flavivirus E polypeptide or a modified flavivirus E polypeptide described herein;(i) a signal sequence, a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second naturally occurring flavivirus NS1Attorney Docket No.: VCCN-016 / 01WO 347597-2099 polypeptide or a modified flavivirus NS1 polypeptide described herein, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(j) a signal sequence, a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, another copy of the first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein with the wing domain substituted with that of a different flavivirus, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(k) a signal sequence, a first copy of a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second copy of the naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(l) a signal sequence, a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a third naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(m) a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(n) a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, another copy of the first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein with the wing domain substituted with that of a different flavivirus, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(o) a first copy of a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second copy of the naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(p) a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a third naturally occurring flavivirus NS1Attorney Docket No.: VCCN-016 / 01WO 347597-2099 polypeptide or a modified flavivirus NS1 polypeptide described herein a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(q) a signal sequence, a purification tag, a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(r) a signal sequence, a purification tag, a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, another copy of the first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein with the wing domain substituted with that of a different flavivirus, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain;(s) a signal sequence, a purification tag, a first copy of a naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second copy of the naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain; or(t) a signal sequence, a purification tag, a first naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a second naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein, a third naturally occurring flavivirus NS1 polypeptide or a modified flavivirus NS1 polypeptide described herein a TM domain described herein, and an ERD or an ESCRT -independent eVLP inducing domain.Linkers

[0221] As noted herein, the modified flavivirus polypeptides described herein may comprise peptide linkers, e.g. to facilitate fusion of the multiple domains or components disclosed herein into a single polypeptide chain. A non-limiting example of a peptide linker is a Glycine-Serine (GS) linker. A GS linker is composed of at least one Glycine and at least one Serine and may vary in length. Non-limiting examples of GS linkers are provided throughout the disclosure and in Table 4A below.

[0222] Table 4A provides exemplary amino acid sequences for flexible linkers.Table 4A, Exemplary flexible linker sequencesAttorney Docket No.: VCCN-016 / 01WO 347597-2099Evaluation of modified flavivirus polypeptides

[0223] Various metrics can be used to determine the suitability of the modified flavivirus polypeptides described herein for use in a vaccine composition. Table 4B provides non-limiting examples of criteria that can be used to for evaluation of the modified flavivirus polypeptides of the disclosure. Non-limiting examples of assays that can be used to assess binding of modified polypeptides to broadly neutralizing or cross-reactive antibodies include enzyme-linked immunosorbent assay (ELISA), flow cytometry, and dot blots. In some embodiments, modified polypeptides that meet at least one of the metrics for advancement are further tested or developed for use in a vaccine composition or other therapeutics. In certain embodiments, modified polypeptides that meet all five metrics for advancement are selected for use. In preferred embodiments, modified polypeptides that meet at least one metric for an ideal target are selected for use.Table 4B: Exemplary selection criteria for advancement of modified polypeptides as vaccine candidatesAttorney Docket No.: VCCN-016 / 01WO 347597-2099Antigen Design

[0224] Disclosed herein are four exemplary antigen designs which can be applied to the modified polypeptides of the present disclosure. The four types of antigen designs are depicted in FIGs. 7A-7D. The modified flavivirus M or NS1 polypeptides described herein are displayed as (1) a chimeric antigen (FIG. 7A), (2) a controlled mosaic (FIG. 7B), (3) a stochastic mosaic (FIG. 7C), or (4) fully orthogonal mixtures (FIG. 7D), each capable of being delivered as a single mRNA vaccine formulation. As an example, FIGs. 7A-7D show the modified flavivirus polypeptides displayed on eVLPs. The four types of antigen designs are also compatible with other forms of antigen delivery, such as in the form of a subunit vaccine.Chimeric design

[0225] Disclosed herein are chimeric M, E, or NS1 proteins, as depicted in FIG. 6A. Chimeric M, E, or NS1 proteins are produced by combining respective sequences from many modified M (with pr deleted), E, or NS1 polypeptides described herein all into a single sequence that captures the diversity of at least two flaviviruses serocomplexes and that can produce mature eVLPs. In some embodiments, the chimeric M, E, or NS1 protein captures the diversity of at least two, at least three, at least four, or at least five flavivirus serocomplexes.Controlled mosaic design

[0226] Also disclosed herein are controlled mosaic E or NS1 proteins, as depicted in FIG. 6B. Controlled mosaic proteins of the disclosure leverage the viral icosahedral surface symmetry to control the mosaic formation to have either 2, 3, or 5 components (based on the 2-fold, 3 -fold, orAttorney Docket No.: VCCN-016 / 01WO 347597-20995-fold symmetries of E proteins on the virion surface). Controlled mosaic proteins are composed of multiple sequences of the modified E or NS1 polypeptides described herein, each of which captures enough sequence diversity predicted to generate broad immune responses against an individual flavivirus serocomplex. In some embodiments, the controlled mosaic proteins comprise two, three, or five modified E or NS1 polypeptide sequences described herein, wherein the sequences of the two, three, or five modified E or NS1 polypeptide sequences are not identical.Stochastic mosaic design

[0227] Also disclosed herein are stochastic mosaic E or NS1 proteins, as depicted in FIG. 6C. Similar to controlled mosaics, stochastic mosaic proteins are composed of multiple sequences of the modified E or NS1 polypeptides described herein, each of which captures enough sequence diversity predicted to generate broad immune responses against an individual flavivirus serocomplex. Unlike controlled mosaics, stochastic mosaics are designed to randomly associate similar E or NS1 polypeptides sequences based on identical interfaces.Orthogonal mixture design

[0228] Also disclosed herein are orthogonal mixtures of E or NSl proteins, as depicted in FIG. 6D. Orthogonal mixtures of protein antigens can be produced by ensuring only specific compositions of modified E or NSl polypeptides are able to associate with one another and assemble as non-mosaic compositions on individual eVLPs, even when co-delivered in a single composition.Enveloped Virus-like particles

[0229] Enveloped virus-like particles are referred to herein as eVLPs. In some embodiments, the modified E, NS1, or prM polypeptides described herein are displayed on the surface of the eVLPs, either in vivo or ex vivo. Without being held to theory or mechanism, VLPs (virus-like particles) are multiprotein structures that mimic the organization and conformation of authentic native viruses but are non-infectious because they do not contain any viral genome. Their production is driven by a viral capsid protein. The eVLPs of the disclosure are non-infectious membraned particles whose production does not require a viral capsid protein and is instead driven by one or more polypeptides that are dependent on host ESCRT machinery, or one or more polypeptides that are entirely independent of host ESCRT machinery (polypeptides comprising ESCRT -independent eVLP inducing domain, including but not limited to amphipathic helices and the platelet-derived growth factor receptor transmembrane domain).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0230] In some embodiments, the one or more polypeptides that are dependent on host ESCRT machinery comprise an ESCRT -recruiting domain (ERD). In some embodiments, the ERD is selected from: (A) viral ERDs, (B) non-human homolog ERDs, (C) synthetic ERDs comprising a backbone capable of forming a coiled-coil structure, also referred to as synthetic coiled-coil ERDs, (D) synthetic ERDs comprising at least two ESCRT -recruiting motifs (ERMs), also referred to as synthetic ERDs with ERMs, and (E) tandem ERDs. In some embodiments, the ERD is derived from flavivirus NS proteins. Examples of polypeptides that depend on host ESCRT machinery to drive eVLPs formation include, but are not limited to, those disclosed in Int. Pub. No. WO2022261230A1 and Int. App. No. PCT / US2025 / 031513, the contents of which are incorporated herein in their entirety by reference. An eVLP whose production is driven by one or more polypeptides that are dependent on host ESCRT machinery are also referred to herein as an ESCRT-driven nanoparticle (EDN). Accordingly, an ERD is also referred to herein as an EDN tag.

[0231] In some embodiments, the one or more polypeptides that are entirely independent of host ESCRT machinery comprises an ESCRT -independent eVLP inducing domain. In some embodiments, the ESCRT -independent eVLP inducing domain is derived from (1) an envelope protein of a virus, or a domain or fragment thereof; (2) a tetraspanin polypeptide, or a domain or fragment thereof, or a binding partner thereof; (3) a lysosome-associated membrane protein 2 (LAMP2) polypeptide, or a domain or fragment thereof, or a binding partner thereof; (4) a a polypeptide that binds to phosphatidylserine, or a domain or fragment thereof; (5) a platelet- derived growth factor receptor (PDGFR) polypeptide, or a domain or fragment thereof; and / or (6) a polypeptide capable of forming an amphipathic helix. In some embodiments, the ESCRT - independent eVLP inducing domain comprises an amphipathic helix or the platelet-derived growth factor receptor transmembrane domain. In some embodiments, the ESCRT -independent eVLP inducing domain comprises the sequence of any one of SEQ ID NOS: 110-112 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. ESCRT -independent eVLP inducing domains are described, for example, in Int. App. No. PCT / US2025 / 039970, the contents of which are incorporated herein in their entirety by reference.

[0232] Table 5A provides amino acid sequences for exemplary ERD sequences that can be incorporated into the modified E, NS1, or prM polypeptides of the present disclosure. In someAttorney Docket No.: VCCN-016 / 01WO 347597-2099 embodiments, the ERD comprises an ESCRT- and ALIX-binding region (EABR) sequence, which facilitates interactions with TSG101 and ALIX, two proteins that are involved in the recruitment of ESCRT machinery to sites of budding. An EABR sequence recruits host ESCRT proteins to the modified E, NS1, or prM polypeptide at the plasma membrane and results in the self-assembly and budding of eVLPs bearing the modified polypeptides described herein.Table 5 A: Amino acid sequences of exemplary ERDsAttorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Table 5B: Amino acid sequences of exemplary signal sequencesAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0233] Disclosed herein are enveloped virus-like particles (eVLPs) that comprise any one or more of the modified flavivirus polypeptides described herein, e.g. the modified flavivirus E polypeptide described herein, the modified NS1 polypeptide described herein, the plurality of modified flavivirus NS1 polypeptide described herein, the modified prM polypeptide described herein, and the plurality of modified flavivirus E polypeptides described herein.

[0234] Disclosed herein are enveloped virus-like particles (eVLPs) that display on their surface any one or more of the modified flavivirus polypeptides described herein, e.g. the modified flavivirus E polypeptide described herein, the modified NS1 polypeptide described herein, the plurality of modified flavivirus NS1 polypeptide described herein, the modified prM polypeptide described herein, and the plurality of modified flavivirus E polypeptides described herein.

[0235] In another aspect, disclosed herein is a cell that comprises any one or more of the modified flavivirus polypeptides described herein, e.g. the modified flavivirus E polypeptide described herein, the modified NS 1 polypeptide described herein, the plurality of modified flavivirus NS1 polypeptide described herein, the modified prM polypeptide described herein, and the plurality of modified flavivirus E polypeptides described herein.

[0236] Also disclosed herein is a cell that displays on its cell surface any one or more of the modified flavivirus polypeptides described herein, e.g. the modified flavivirus E polypeptide described herein, the modified NS 1 polypeptide described herein, the plurality of modified flavivirus NS1 polypeptide described herein, the modified prM polypeptide described herein, and the plurality of modified flavivirus E polypeptides described herein.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0237] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human mammalian cell. In some embodiments, the cell is an immortalized cell.Amino acid sequences for various constructs and components

[0238] Table 6A provides exemplary amino acid sequences comprising the modified E, NS1, and / or prM polypeptides described herein, as used in various constructs described herein. A description is also provided to indicate the polypeptides or domains included in each construct. Unless otherwise indicated, sequences for E protein (full length or truncated) are in bold, sequences for M protein (continuous or separated by insertions) are underlined, and "pr" sequences are italicized. A leading “m” denotes a methionine encoded by an artificial start codon. Although the sequences of Table 6A may include linkers, these linkers are optional in nature and may be omitted or replaced by other suitable linkers.Table 6 A: Exemplary amino acid sequences of constructsAttorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0239] Table 6B and Table 6C provide additional exemplary constructs described herein comprising a modified E, NS1, and / or prM polypeptides of the disclosure. A description of each construct is provided to indicate the polypeptides or domains included in each construct. Although the sequences of Table 6B and Table 6C may include linkers, these linkers are optional in nature and may be omitted or replaced by other suitable linkers.Table 6B: Exemplary constructsAttorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Table 6C: Exemplary constructsAttorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0240] Table 6D below provides amino acid sequences of exemplary transmembrane (TM) domains for use in the modified flavivirus polypeptides of the disclosure.Table 6D: Exemplary transmembrane domainsAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0241] Table 6E below provides amino acid sequences of exemplary transmembrane (TM) domains for use in the modified flavivirus polypeptides of the disclosure.Table 6E: Exemplary signal sequences

[0242] Table 6F below provides amino acid sequences of exemplary endocytosis prevention motifs (EPM) for use in the modified flavivirus polypeptides of the disclosure.Table 6F: Exemplary endocytosis prevention motifsAttorney Docket No.: VCCN-016 / 01WO 347597-2099Polynucleotides

[0243] In another aspect, also disclosed herein are polynucleotides that encode the amino acid sequence of the modified flavivirus polypeptides of the disclosure including the modified E polypeptides, the modified NS1 polypeptides, the plurality of modified flavivirus NS1 polypeptides, the modified prM polypeptides, and the pluralities of modified flavivirus E polypeptides of the present disclosure. In some embodiments, the polynucleotide is or comprises DNA. In some embodiments, the polynucleotide is or comprises RNA. In some embodiments, the polynucleotide is or comprises mRNA.

[0244] In some embodiments, the polynucleotides of the present disclosure may comprise a promoter. In some embodiments, the promoter comprises a ubiquitous promoter, an inducible promoter, a tissue-specific promoter and / or a lineage-specific promoter. In some embodiments, the ubiquitous promoter is selected from the group consisting of: a viral simian virus 40 (SV40) (e.g., early or late), a Rous sarcoma virus (RSV) LTR, an RSV promoter, a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a Moloney murine leukemia virus (MoMLV) LTR promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, HS, P7.S, and Pl 1 promoters from vaccinia virus, an elongation factor I-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3- phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein S (HSP AS), heat shock protein 90 kDa beta, member 1 (HSP90B 1 ), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-I (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CB A) promoter, a CAG promoter, a CASI promoter, a CBH promoter, or any combination thereof.

[0245] In some embodiments, the polynucleotides of the present disclosure are operably linked to a tandem gene expression element (e.g., an internal ribosomal entry site (IRES), foot-and- mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), poly A tails or any combination thereof. In some embodiments, the polynucleotide(s) encoding fusion protein(s) comprises a transcript stabilization element (e.g., woodchuck hepatitis post-translationalAttorney Docket No.: VCCN-016 / 01WO 347597-2099 regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof.

[0246] In some embodiments, the polynucleotides of the present disclosure may include poly-A tails. Inclusion of a 3’ poly(A) tail in an mRNA sequence of the disclosure can contribute to the stability and translation efficiency of the mRNA. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression.

[0247] In some embodiments, where the polynucleotides of the present disclosure comprise mRNA, the mRNA comprises a poly(A) sequence having at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and / or a cap. The nucleoside sequence in an mRNA molecule provides instructions that cells use to create specific proteins. mRNA is a molecule that typically is composed of four different nucleosides: adenosine, guanosine, cytidine, and uridine. Vaccines containing mRNA can trigger the body’s own immune system to attack the mRNA molecule. This immune response may destroy the mRNA before it can have its intended effect. Different modifications to the mRNA molecule have been developed to disguise the mRNA from the body’s immune system, such as those described in U.S. Patent No. 10,898,574, U.S. Patent No. 10,703,789, U.S. Patent No. 10,577,403, and U.S. Patent No. 10, 064,959, the contents of which are herein incorporated by reference in their entirety. In some embodiments, where the polynucleotides of the present disclosure comprise mRNA, the mRNA comprises one or more modified nucleotides selected from the group consisting of: pseudouridine, N-l-methyl-pseudouridine, 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5 -iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine,0(6)-methylguanine, and 2-thiocytidine. In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments, the modified nucleoside is selected from pseudouridine (y), N 1-methyl-pseudouridine (m ly), and 5-methyl-uridine(m5U). In some embodiments, the mRNA comprises a modified nucleotide inAttorney Docket No.: VCCN-016 / 01WO 347597-2099 place of one or more uridines. In some embodiments where the polynucleotides comprise mRNA, the mRNA may be formulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA may be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating mRNA.

[0248] In some embodiments, where the polynucleotide of the present disclosure is mRNA, the mRNA molecule can be synthesized through in vitro transcription of a corresponding DNA template molecule. For example, synthetic mRNA can be produced by in vitro transcription of a cDNA template, such as plasmid DNA (pDNA).Vectors

[0249] Disclosed herein are vectors comprising the polynucleotides described herein or a plurality of the polynucleotides thereof. In some embodiments, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof. In an exemplary embodiment, the vector is an LNP comprising an mRNA polynucleotide. Generally, LNP has four components: ionizable cationic lipids, phospholipids, cholesterol, and PEG lipids. Each component contributes to LNP stability, transfection efficacy, and safety.

[0250] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, adenovirus vector, modified vaccinia virus Ankara (MV A) vector, poxvirus vector, or Vesicular stomatitis virus (VSV) vector. In certain embodiments wherein the viral vector is an adenovirus vector, the adenovirus is Ad26, Ad34, Ad35, Ad48, or Ad5HVR48. Vectors of the present disclosure may be formulated in a lipid nanoparticle (LNP). In some embodiments, the vectors may be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating the polynucleotides.

[0251] In some embodiments, the polynucleotides and vectors of the present disclosure may be administered to a subject as a vaccine composition to prevent a disease or disorder, as will be described in more detail herein.Vaccine Compositions

[0252] Disclosed herein are vaccine compositions comprising the modified E polypeptide described herein, the modified NS 1 polypeptide described herein, the plurality of modified NS1Attorney Docket No.: VCCN-016 / 01WO 347597-2099 polypeptides described herein, the modified prM polypeptide described herein, the plurality of modified E polypeptides described herein, the eVLP described herein, the polynucleotide described herein, or the vector described herein. The modified E, NS1, and prM polypeptides described herein may be chimeric. A plurality of these modified polypeptides may self-assemble into controlled mosaics, stochastic mosaics, or orthogonal mosaics. In some embodiments, the vaccine compositions described herein comprises modified polypeptides or a plurality thereof that are chimeric or have assembled into controlled mosaics, stochastic mosaics, or orthogonal mosaics.

[0253] In some embodiments, the vaccine composition comprises the polynucleotides of the vectors described herein. In some embodiments, the vaccine composition comprises one or more adjuvants. Suitable adjuvants include, but are not limited to, aluminum salts, MF59, PLA, PGLA, polyinosinic:polycytidylic acid (Poly(I:C)), Monophosphoryl lipid A (MPL), Imiquimod (R837; l-(2-methylpropyl)-lH-imidazo [4,5-c]quinolin-4-amine), resiquimod (R848, 4-amino- 2-(etoximetil)-a,a-dimethyl-lH-imidazo [4,5-c]quinoline-l-ethanol), CpG ODNs, AS01, AS01B, AS02, AS03, AS04, and Matrix-M. The vaccine composition can be formulated with any pharmaceutically acceptable carrier, excipient, or diluent. Suitable carriers, excipients, or diluents include, but are not limited to, sodium chloride, monobasic sodium phosphate, dibasic sodium phosphate, potassium phosphate, monobasic potassium phosphate, potassium chloride, calcium chloride, sodium taurodeoxycholate, ovalbumin, sucrose, neomycin sulfate, polymyxin B, betapropiolactone, hydrocortisone, thimerosal, sorbitol, gelatin, sucrose, D-mannose, D- fructose, dextrose, human serum albumin, plasdone C, anhydrous lactose, microcrystalline cellulose, polacrilin potassium, magnesium stearate, cellulose acetate phthalate, alcohol, acetone, and castor oil.

[0254] The vaccine compositions described herein may offer broad protection against infection by multiple flaviviruses and / or reduce ADE. The vaccine compositions may reduce the severity of one or more symptoms of a flavivirus infection.Evaluation of vaccine candidates

[0255] Various metrics assessing the antigens of the vaccine compositions described herein can be used to determine the efficacy of the vaccine compositions, including the platform and / or adjuvants used. For example, a vaccine candidate may be selected for advancement if a decrease in NSl-mediated endothelial cell disruption compared to naturally occurring flavivirus proteins, as measured by in vitro endothelial permeability assays, is observed. Table 7 provides nonAttorney Docket No.: VCCN-016 / 01WO 347597-2099 limiting examples of criteria that can be used for candidate evaluation and corresponding metrics that should be or are ideally met for vaccine candidate advancement.Table 7: Selection criteria for vaccine candidatesMethods

[0256] Also disclosed herein are methods of inducing anti-flavivirus antibodies, inducing a flavivirus-specific immune response, preventing a flavivirus infection, and treating a flavivirus infection or a symptom thereof in a subject having, or at risk of having, a flavivirus infection. In some embodiments, the methods comprise administering to the subject in need thereof an effective amount of any one of the modified E polypeptide described herein, the modified NS1 polypeptide described herein, a plurality of the modified E polypeptides described herein, a plurality of the modified NS1 polypeptides described herein, the eVLP comprising any of the modified polypeptides described herein, the polynucleotide encoding any of the modified polypeptides or the eVLP described herein, the vector comprising the polynucleotides describedAttorney Docket No.: VCCN-016 / 01WO 347597-2099 herein, or the vaccine composition described herein. In some embodiments, the anti-flavivirus antibodies that are induced target M protein, E protein and / or NS1 protein. In certain embodiments, the anti-flavivirus antibodies are broadly neutralizing. In some embodiments, the method comprises administering to the subject in need thereof at least a first dose of an effective amount of any one of the polypeptides of the disclosure, the polynucleotides of the disclosure, the eVLPs of the disclosure, the vectors of the disclosure, or the vaccine compositions of the disclosure.

[0257] In some embodiments, the administration is in the context of vaccination, and used for the prevention of a flavivirus infection. In some embodiments, the administration is in the context of treatment, and used for the mitigation of an already existing flavivirus infection and facilitates antibody -mediated clearance of the infection. In some embodiments, the administration is in the context of treatment and used for the prophylactical treatment to prevent infection with a flavivirus or protect against a disease or symptom thereof caused by flavivirus infection. In some embodiments, vaccination or treatment provides partial protection against a flavivirus infection. In some embodiments, vaccination or treatment provides full protection against a flavivirus infection.

[0258] In some embodiments, the subject has, or is at risk of having, an infection of a flavivirus. In certain embodiments, the subject has, or is at risk of having, an infection of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0259] In some embodiments, the methods of treating or preventing a disease or disorder in a subject in need thereof may include administering to the subject one or more additional doses of an effective amount of any one of the modified E polypeptide described herein, the modified NS1 polypeptide described herein, a plurality of the modified E polypeptides described herein, a plurality of the modified NS1 polypeptides described herein, the eVLP comprising any of the modified polypeptides described herein, the polynucleotide encoding any of the modified polypeptides or the eVLP described herein, the vector comprising the polynucleotides described herein, or the vaccine composition described herein. In some embodiments, administering to the subject an additional dose may include administering to the subject at least a second dose of an effective amount of any one of the modified E polypeptide described herein, the modified NS1 polypeptide described herein, a plurality of the modified E polypeptides described herein, aAttorney Docket No.: VCCN-016 / 01WO 347597-2099 plurality of the modified NS1 polypeptides described herein, the eVLP comprising any of the modified polypeptides described herein, the polynucleotide encoding any of the modified polypeptides or the eVLP described herein, the vector comprising the polynucleotides described herein, or the vaccine composition described herein.

[0260] The route of administration may be selected from any known method suitable for the treatment. Suitable routes of administration include, but are not limited to, parenteral delivery, such as intramuscular, intradermal, subcutaneous, intramedullary injections, as well as, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. In some embodiments, the route of administration is intramuscular. In some embodiments, the route of administration is intravenous. In some embodiments, the route of administration is subcutaneous. In some embodiments, the route of administration may include injection, inhalation or by oral, parenteral or rectal administration. In some embodiments, the route of administration may use traditional syringes and needleless injection devices. For injection, the effective amount of any one of the modified E polypeptide described herein, the modified NS1 polypeptide described herein, a plurality of the modified E polypeptides described herein, the plurality of the modified NS1 polypeptides described herein, the eVLP comprising any of the modified polypeptides described herein, the polynucleotide encoding any of the modified polypeptides or the eVLP described herein, the vector comprising the polynucleotides described herein, or the vaccine composition described herein can be formulated in aqueous solutions, optionally in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. In some embodiments, administering comprises aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intraci sternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof. In some embodiments, administering comprises intramuscular injection of any one or more of: the modified flavivirus E polypeptides of the disclosure, the modified NS1 polypeptides of the disclosure, the plurality of modified flavivirus NS1 polypeptides of the disclosure, the modified prM polypeptides of the disclosure, the plurality of modified flavivirus E polypeptides of the disclosure, the polynucleotides of the disclosure, the vectors of the disclosure, the cells of the disclosure, the eVLPs of the disclosure, and the vaccine compositions of the disclosure.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0261] In some embodiments, the subject is a mammal. Non-limiting examples of mammals include, a human, a non-human primate, a rodent, a dog, a cat, a rabbit, a cow, a horse, a goat, a sheep, a llama, a camel, a donkey, a bat, a deer, a bear, a squirrel, or a pig. In some embodiments, the subject is a human subject. In some embodiments, the subject in need thereof is not a human subject. In some embodiments, the subject is a bird, such as a chicken, a duck, a pheasant, a turkey, or a goose. In some embodiments, the biological sex of the subject is female. In some embodiments, the subject is pregnant. In some embodiments, the subject being treated in accordance with the methods described herein has been diagnosed with a flavivirus infection or is at risk of being infected by a flavivirus. In some embodiments, the subject has had one or more prior infections with a flavivirus. In some embodiments, the subject has never been infected with a flavivirus.

[0262] Also disclosed herein are the modified flavivirus E polypeptides of the disclosure, the modified NS1 polypeptides of the disclosure, the plurality of modified flavivirus NS1 polypeptides of the disclosure, the modified prM polypeptides of the disclosure, the plurality of modified flavivirus E polypeptides of the disclosure, the polynucleotides of the disclosure, the vectors of the disclosure, the cells of the disclosure, the eVLPs of the disclosure, or the vaccine compositions of the disclosure for use in the treatment of a flavivirus infection or a symptom thereof. In some embodiments, the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). In some embodiments, the modified flavivirus E polypeptides of the disclosure, the modified NS1 polypeptides of the disclosure, the plurality of modified flavivirus NS1 polypeptides of the disclosure, the modified prM polypeptides of the disclosure, the plurality of modified flavivirus E polypeptides of the disclosure, the polynucleotides of the disclosure, the vectors of the disclosure, the cells of the disclosure, the eVLPs of the disclosure, or the vaccine compositions of the disclosure is delivered through aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracistemal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof.

[0263] Also disclosed herein is a use of the modified flavivirus E polypeptides of the disclosure, the modified NS1 polypeptides of the disclosure, the plurality of modified flavivirus NS1Attorney Docket No.: VCCN-016 / 01WO 347597-2099 polypeptides of the disclosure, the modified prM polypeptides of the disclosure, the plurality of modified flavivirus E polypeptides of the disclosure, the polynucleotides of the disclosure, the vectors of the disclosure, the cells of the disclosure, the eVLPs of the disclosure, or the vaccine compositions of the disclosure in the manufacture of a medicament for the treatment of a flavivirus infection or a symptom thereof. In some embodiments, the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV). In some embodiments, the medicament is delivered through aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracistemal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof.

[0264] Symptoms of a flavivirus infection include, but are not limited to, fever, headache, body aches, joint pain, rash, vomiting, diarrhea, conjunctivitis, febrile illness, arthralgia, myalgia, organic damage, hemorrhagic disease, hemorrhagic fever, and neuroinvasive disease. Nonlimiting examples of neuroinvasive disease include meningitis, encephalitis, and paralysis.

[0265] The disclosure and the embodiments described herein are further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference for all purposes.ENUMERATED EMBODIMENTS

[0266] Provided herein are non-limiting exemplary enumerated embodiments.

[0267] Embodiment 1-1. A modified flavivirus E polypeptide comprising at least one modification relative to a wild-type E polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

[0268] Embodiment 1-2. The modified flavivirus E polypeptide of embodiment I- 1, wherein the characteristic is increased stability, increased immunogenicity, improved breadth of immunogenicity, increased secretion as enveloped virus-like particles (eVLPs), improved ability to form a lattice, improved ability to make mosaics, improved ability to make mosaic lattices, improved expression, or decreased potential to induce antibody-dependent enhancement (ADE).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0269] Embodiment 1-3. The modified flavivirus E polypeptide of embodiment 1-1 or 1-2, wherein the modified polypeptide exhibits increased stability in an acidic environment or at physiological temperatures.

[0270] Embodiment 1-4. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-3, wherein the at least one modification is ablation of an immunodominant ADE-inducing epitope within the E fusion loop and whereby the modified polypeptide exhibits decreased potential to induce ADE.

[0271] Embodiment 1-5. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-4, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0272] Embodiment 1-6. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-5, wherein the at least one modification comprises an amino acid deletion.

[0273] Embodiment 1-7. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-6, wherein the at least one modification comprises an amino acid substitution.

[0274] Embodiment 1-8. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-7, wherein the at least one modification comprises an amino acid insertion.

[0275] Embodiment 1-9. The modified flavivirus E polypeptide of embodiment 1-8, wherein the amino acid insertion comprises insertion of an endoplasmic reticulum (ER) signal sequence, a portion of a flavivirus nonstructural (NS) protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a potential N-linked glycosylation site (PNGS) or a combination thereof.

[0276] Embodiment I- 10. The modified flavivirus E polypeptide of embodiment 1-9, wherein the amino acid insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5.

[0277] Embodiment 1-11. The modified flavivirus E polypeptide of embodiment 1-9 or I- 10, wherein the flavivirus nonstructural protein is NS2 or NS3.

[0278] Embodiment 1-12. The modified flavivirus E polypeptide of any one of embodiments 1-9 to 1-11, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD.

[0279] Embodiment 1-13. The modified flavivirus E polypeptide of embodiment 1-12, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain and wherein amino acid sequence of the transmembrane domain comprises the amino acid sequenceAttorney Docket No.: VCCN-016 / 01WO 347597-2099 of the TM sequence of any one of SEQ ID NOs: 16 to 30 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto.

[0280] Embodiment 1-14. The modified flavivirus E polypeptide of embodiment 1-9, wherein the insertion comprises an ER export sequence and wherein the export sequence comprises the YTDIEM motif.

[0281] Embodiment 1-15. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-14, wherein the at least one modification is relative to a wild-type flavivirus E protein sequence.

[0282] Embodiment 1-16. The modified flavivirus E polypeptide of embodiment 1-15, wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), or Murray Valley Encephalitis virus (MVEV).

[0283] Embodiment 1-17. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-16, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 1 to 17 or a truncated sequence thereof.

[0284] Embodiment 1-18. A modified NS1 polypeptide comprising at least one modification relative to a wild-type NS1 polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic, wherein the characteristic is increased stability, increased immunogenicity, decrease in capacity to trigger endothelial barrier permeability, improved ability to form stable oligomers for display in various forms, or increased breadth of the resultant immune response.

[0285] Embodiment 1-19. The modified flavivirus NS1 polypeptide of embodiment 1-18, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0286] Embodiment 1-20. The modified flavivirus NS1 polypeptide of embodiment 1-18 or 1-19, wherein the at least one modification comprises an amino acid deletion.

[0287] Embodiment 1-21. The modified flavivirus NS1 polypeptide of any one of embodiments 1-18 to 1-20, wherein the at least one modification comprises an amino acid substitution.

[0288] Embodiment 1-22. The modified flavivirus NS1 polypeptide of any one of embodiments 1-18 to 1-21, wherein the at least one modification comprises an amino acid insertion.

[0289] Embodiment 1-23. The modified flavivirus NS1 polypeptide of embodiment 1-22, wherein the amino acid insertion comprises insertion of an ER signal sequence, a portion of aAttorney Docket No.: VCCN-016 / 01WO 347597-2099 flavivirus NS protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT- independent eVLP inducing domain, or a combination thereof.

[0290] Embodiment 1-24. The modified flavivirus NS1 polypeptide of embodiment 1-23, wherein the insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5.

[0291] Embodiment 1-25. The modified flavivirus NS1 polypeptide of embodiment 1-23 or 1-24, wherein the flavivirus nonstructural protein is NS2 or NS3.

[0292] Embodiment 1-26. The modified flavivirus NS1 polypeptide of any one of embodiments 1-23 to 1-25, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD.

[0293] Embodiment 1-27. The modified flavivirus NS1 polypeptide of embodiment 1-26, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain, optionally wherein the transmembrane domain comprises the amino acid sequence of the TM sequence of any one of SEQ ID NOs: 16 to 30 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto.

[0294] Embodiment 1-28. The modified flavivirus NS1 polypeptide of embodiment 1-23, wherein the insertion comprises an ER export sequence and wherein the export sequence comprises the YTDIEM motif

[0295] Embodiment 1-29. The modified flavivirus NS1 polypeptide of any one of embodiments 1-18 to 1-28, wherein the at least one modification is relative to a wild-type NS1 protein from a flavivirus of the Flavirvirus genus.

[0296] Embodiment 1-30. The modified flavivirus NS1 polypeptide of embodiment 1-29, wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0297] Embodiment 1-31. The modified flavivirus NS1 polypeptide of any one of embodiments 1-18 to 1-30, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 20 to 34.

[0298] Embodiment 1-32. A plurality of modified flavivirus NS1 polypeptides selected from any one of embodiments 1-18 to 1-31, wherein the plurality of NS1 polypeptides are fused into a single polypeptide chain, and wherein the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides with identical or unique sequences.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0299] Embodiment 1-33. The plurality of modified flavivirus NS1 polypeptides of embodiment 1-32, wherein the plurality of NS1 polypeptides comprises two or more modified NS1 polypeptides with unique sequences, thereby improving the breadth of immunogenicity.

[0300] Embodiment 1-34. A modified flavivirus prM polypeptide comprising at least one modification relative to a wild-type prM polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

[0301] Embodiment 1-35. The modified flavivirus prM polypeptide of embodiment 1-34, wherein the characteristic is the increased ability to stabilize the conformation of wild-type or modified flavivirus E, improved expression, improved secretion, a decrease in immature, conformationally heterogeneous virions, or a combination thereof.

[0302] Embodiment 1-36. The modified flavivirus prM polypeptide of embodiment 1-34 or 1-35, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0303] Embodiment 1-37. The modified flavivirus prM polypeptide of any one of embodiments 1-34 to 1-36, wherein the at least one modification comprises an amino acid deletion.

[0304] Embodiment 1-38. The modified flavivirus prM polypeptide of any one of embodiments 1-34 to 1-37, wherein the at least one modification comprises an amino acid substitution.

[0305] Embodiment 1-39. The modified flavivirus prM polypeptide of any one of embodiments 1-34 to 1-38, wherein the at least one modification comprises an amino acid insertion.

[0306] Embodiment 1-40. The modified flavivirus prM polypeptide of embodiment 1-36, wherein the amino acid deletion comprises deletion of the pr amino acid sequence or a portion thereof, optionally wherein the pr amino acid sequence comprises the sequence of any one of SEQ ID NOs: 70-77 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto.

[0307] Embodiment 1-41. The modified flavivirus E polypeptide of any one of embodiments 1-1 to 1-17, further fused to a second flavivirus polypeptide.

[0308] Embodiment 1-42. The modified flavivirus E polypeptide of embodiment 1-41, wherein the second polypeptide comprises a wild-type prM or the modified flavivirus prM polypeptide of any one of embodiments 1-32 to 1-40, the NS1 polypeptide of any one of embodiments 1-18 to I- 31, and / or the plurality of NS1 polypeptides of embodiment 1-32 or 1-33.

[0309] Embodiment 1-43. The modified flavivirus E polypeptide of embodiment 1-42, wherein the second polypeptide comprises a wild-type prM or the modified flavivirus prM polypeptide of any one of embodiments 1-32 to 1-40, optionally wherein the second polypeptide is fused to the N- or C-terminus of the modified E polypeptide.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0310] Embodiment 1-44. A plurality of modified flavivirus E polypeptides selected from any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, wherein the plurality of polypeptides are fused into a single chimeric polypeptide chain.

[0311] Embodiment 1-45. The plurality of modified flavivirus E polypeptides of embodiment I- 44, wherein the plurality of polypeptides assembles into a mosaic or mosaic lattice.

[0312] Embodiment 1-46. The plurality of modified flavivirus E polypeptides of embodiment I- 44 or 1-45, comprising two, three, or five unique modified E polypeptides selected from any one of embodiments 1-1 to 1-17 and 1-41 to 1-43, optionally wherein the modified E polypeptides have orthogonal interfaces.

[0313] Embodiment 1-47. The plurality of modified flavivirus E polypeptides of embodiment I- 44, wherein the plurality of polypeptides assemble into a stochastic mosaic, optionally comprising six, seven, or eight unique modified E polypeptides with identical interfaces.

[0314] Embodiment 1-48. An enveloped virus-like particle (eVLP) comprising the modified flavivirus E polypeptide of any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified flavivirus NS1 polypeptide of embodiment 1-32 or 1-33, the modified prM polypeptide of any one of embodiments 1-34 to 1-40, or the plurality of modified flavivirus E polypeptides of any one of embodiments 1-44 to 1-47.

[0315] Embodiment 1-49. A polynucleotide encoding the amino acid sequence of the modified E polypeptide of any one of embodiments 1-1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified flavivirus NS1 polypeptide of embodiment 1-32 or 1-33, the modified prM polypeptide of any one of embodiments 1-34 to 1-40, or the plurality of modified flavivirus E polypeptides of any one of embodiments 1-44 to 1-47.

[0316] Embodiment 1-50. The polynucleotide of embodiment 1-49, wherein the polynucleotide comprises DNA.

[0317] Embodiment 1-51. The polynucleotide of embodiment 1-49, wherein the polynucleotide comprises RNA.

[0318] Embodiment 1-52. The polynucleotide of embodiment 1-51, wherein the polynucleotide comprises mRNA.

[0319] Embodiment 1-53. The polynucleotide of embodiment 1-52, wherein the polynucleotide comprises modified nucleosides.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0320] Embodiment 1-54. A vector comprising the polynucleotide of any one of embodiments I- 49 to 1-53 or a plurality of polynucleotides selected from any one of embodiments 1-49 to 1-53.

[0321] Embodiment 1-55. The vector of embodiment 1-54, wherein the vector is a viral vector.

[0322] Embodiment 1-56. The vector of embodiment 1-55, wherein the viral vector is an adeno- associated virus (AAV) vector, adenovirus vector, modified vaccinia virus Ankara (MV A) vector, poxvirus vector, or Vesicular stomatitis virus (VSV) vector.

[0323] Embodiment 1-57. The vector of embodiment 1-56, wherein the adenovirus vector is Ad26, Ad34, Ad35, Ad48, or Ad5HVR48.

[0324] Embodiment 1-58. The vector of embodiment 1-54, wherein the vector is a non-viral vector.

[0325] Embodiment 1-59. The vector of embodiment 1-58, wherein the non-viral vector is a plasmid.

[0326] Embodiment 1-60. The vector of embodiment 1-58, wherein the non-viral vector is a lipid nanoparticle (LNP).

[0327] Embodiment 1-61. A vaccine composition comprising the modified E polypeptide of any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified NS1 polypeptides of embodiment 1-32 or I- 33, the modified prM polypeptide of any one of embodiments 1-34 to 1-40, the plurality of modified E polypeptides of any one of embodiments 1-44 to 1-47, the eVLP of embodiment 1-48, the polynucleotide of any one of embodiments 1-49 to 1-53, or the vector of any one of embodiments 1-54 to 1-60.

[0328] Embodiment 1-62. The vaccine composition of embodiment 1-61, further comprising one or more adjuvants.

[0329] Embodiment 1-63. The vaccine composition of embodiment 1-61 or 1-62, further comprising one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0330] Embodiment 1-64. A kit comprising the modified E polypeptide of any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified NS1 polypeptides of embodiment 1-32 or I- 33, the modified prM polypeptide of any one of embodiments 1-34 to 1-40, the plurality of modified E polypeptides of any one of embodiments 1-44 to 1-47, the eVLP of embodiment 1-48, the polynucleotide of any one of embodiments 1-49 to 1-53, the vector of any one of embodiments 1-54 to 1-60, or the vaccine composition of any one of embodiments 1-61 to 1-63, and instructions for use.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0331] Embodiment 1-65. A method of preventing a flavivirus infection or a symptom thereof in a subject having, or at risk of having, a flavivirus infection, comprising administering to the subject an effective amount of the modified E polypeptide of any one of embodiments 1-1 to I- 17 and 1-41 to 1-43, the modified NS 1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified flavivirus NS1 polypeptide of embodiment 1-32 or 1-33, the plurality of modified E polypeptides of any one of embodiments 1-44 to 1-47, the eVLP of embodiment 1-48, the polynucleotide of any one of embodiments 1-49 to 1-53, the vector of any one of embodiments 1-54 to 1-60, or the vaccine composition of any one of embodiments 1-61 to 1-63.

[0332] Embodiment 1-66. The method of preventing a flavivirus infection of embodiment 1-65, wherein the subject has, or is at risk of having, an infection of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0333] Embodiment 1-67. The method of preventing a flavivirus infection of embodiment 1-65 or 1-66, wherein the subject is a mammal or a bird, optionally wherein the subject is pregnant.

[0334] Embodiment 1-68. The method of preventing a flavivirus infection of embodiment 1-67, wherein the subject is a mammal.

[0335] Embodiment 1-69. The method of preventing a flavivirus infection of embodiment 1-68, wherein the subject is a human, optionally wherein the human subject is pregnant.

[0336] Embodiment 1-70. The method of preventing a flavivirus infection of embodiment 1-67, wherein the subject is a bird.

[0337] Embodiment 1-71. A method of treating a flavivirus infection or a symptom thereof in a subject having a flavivirus infection, comprising administering to the subject an effective amount of the modified E polypeptide of any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified flavivirus NS1 polypeptide of embodiment 1-32 or 1-33, the plurality of modified E polypeptides of any one of embodiments 1-44 to 1-47, the eVLP of embodiment 1-48, the polynucleotide of any one of embodiments 1-49 to 1-53, the vector of any one of embodiments 1-54 to 1-60, or the vaccine composition of any one of embodiments 1-61 to 1-63.

[0338] Embodiment 1-72. A method of inducing anti -flavivirus antibodies in a subject, comprising administering to the subject an effective amount of the modified E polypeptide of any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any oneAttorney Docket No.: VCCN-016 / 01WO 347597-2099 of embodiments 1-18 to 1-31, the plurality of modified flavivirus NS1 polypeptide of embodiment 1-32 or 1-33, the plurality of modified E polypeptides of any one of embodiments I- 44 to 1-47, the eVLP of embodiment 1-48, the polynucleotide of any one of embodiments 1-49 to 1-53, the vector of any one of embodiments 1-54 to 1-60, or the vaccine composition of any one of embodiments 1-61 to 1-63.

[0339] Embodiment 1-73. The method of embodiment 1-72, wherein the anti -flavivirus antibodies target E protein and / or NS1 protein.

[0340] Embodiment 1-74. A method for inducing a flavivirus-specific immune response, comprising administering to the subject an effective amount of the modified E polypeptide of any one of embodiments I- 1 to 1-17 and 1-41 to 1-43, the modified NS1 polypeptide of any one of embodiments 1-18 to 1-31, the plurality of modified flavivirus NS1 polypeptide of embodiment 1-32 or 1-33, the plurality of modified E polypeptides of any one of embodiments I- 44 to 1-47, the eVLP of embodiment 1-48, the polynucleotide of any one of embodiments 1-49 to 1-53, the vector of any one of embodiments 1-54 to 1-60, or the vaccine composition of any one of embodiments 1-61 to 1-63, optionally wherein the immune response provides immune protection for at least two flaviviruses.

[0341] Embodiment II- 1. A modified flavivirus E polypeptide comprising at least one modification relative to a naturally occurring E polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

[0342] Embodiment II-2. The modified flavivirus E polypeptide of embodiment II- 1, wherein the at least one improved characteristic comprises increased stability, increased immunogenicity, improved breadth of immunogenicity, increased secretion as enveloped virus-like particles (eVLPs), improved ability to form a lattice, improved ability to make mosaics, improved ability to make mosaic lattices, improved expression, or decreased potential to induce antibodydependent enhancement (ADE).

[0343] Embodiment II-3. The modified flavivirus E polypeptide of embodiment II- 1 or II-2, wherein the modified polypeptide exhibits increased stability in an acidic environment or at physiological temperatures.

[0344] Embodiment II-4. The modified flavivirus E polypeptide of any one of embodiments II- 1 to II-3, wherein the at least one modification is ablation of an immunodominant ADE-inducing epitope within the E fusion loop and whereby the modified polypeptide exhibits decreased potential to induce ADE.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0345] Embodiment II-5. The modified flavivirus E polypeptide of any one of embodiments II- 1 to II-4, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0346] Embodiment II-6. The modified flavivirus E polypeptide of any one of embodiments II- 1 to II-5, wherein the at least one modification comprises an amino acid deletion.

[0347] Embodiment II-7. The modified flavivirus E polypeptide of any one of embodiments II- 1 to II-6, wherein the at least one modification comprises an amino acid substitution.

[0348] Embodiment II-8. The modified flavivirus E polypeptide of any one of embodiments II- 1 to II-7, wherein the at least one modification comprises an amino acid insertion.

[0349] Embodiment II-9. The modified flavivirus E polypeptide of embodiment II-8, wherein the amino acid insertion comprises insertion of an endoplasmic reticulum (ER) signal sequence, a portion of a flavivirus nonstructural (NS) protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a potential N-linked glycosylation site (PNGS) or a combination thereof.

[0350] Embodiment II- 10. The modified flavivirus E polypeptide of embodiment II-9, wherein the amino acid insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5.

[0351] Embodiment II- 11. The modified flavivirus E polypeptide of embodiment II-9 or 11-10, wherein the flavivirus nonstructural protein is NS2 or NS3.

[0352] Embodiment 11-12. The modified flavivirus E polypeptide of any one of embodiments II- 9 to II- 11, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD.

[0353] Embodiment 11-13. The modified flavivirus E polypeptide of any one of embodiments II-9 to 11-12, the amino acid insertion comprises insertion of an ERD, optionally wherein the ERD comprises the sequence of any one of SEQ ID NOS: 350-387 or a sequence having at least 70% sequence identity thereto.

[0354] Embodiment 11-14. The modified flavivirus E polypeptide of embodiment II-9, wherein the insertion comprises an ER export sequence and wherein the export sequence comprises the YTDIEM motif (SEQ ID NO: 144).

[0355] Embodiment 11-15. The modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-14, wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virusAttorney Docket No.: VCCN-016 / 01WO 347597-2099(POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), or Murray Valley Encephalitis virus (MVEV).

[0356] Embodiment 11-16. The modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-15, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 1-17 or a truncated sequence thereof.

[0357] Embodiment 11-17. A modified NS1 polypeptide comprising at least one modification relative to a naturally occurring NS1 polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic, wherein the characteristic is increased stability, increased immunogenicity, decrease in capacity to trigger endothelial barrier permeability, improved ability to form stable oligomers for display in various forms, or increased breadth of the resultant immune response.

[0358] Embodiment 11-18. The modified flavivirus NS1 polypeptide of embodiment 11-17, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0359] Embodiment 11-19. The modified flavivirus NS1 polypeptide of embodiment 11-17 or Ills, wherein the at least one modification comprises an amino acid deletion.

[0360] Embodiment 11-20. The modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-19, wherein the at least one modification comprises an amino acid substitution.

[0361] Embodiment 11-21. The modified flavivirus NS1 polypeptide of embodiment 11-20, wherein the amino acid substitution comprises substitution of an amino acid at a position corresponding to position 115, 118, 119, or 207 of SEQ ID NO: 26.

[0362] Embodiment 11-22. The modified flavivirus NS1 polypeptide of embodiment 11-20 or II- 21, wherein the amino acid substitution comprises substitution of an amino acid at a position corresponding to position 115 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A).

[0363] Embodiment 11-23. The modified flavivirus NS1 polypeptide of any one of embodiments 11-20 to 11-22, wherein the amino acid substitution comprises:

[0364] (a) substitution of an amino acid at a position corresponding to position 118 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A);

[0365] (b) substitution of an amino acid at a position corresponding to position 119 of SEQ ID NO: 26, wherein the amino acid substitution is Glycine (G) to Alanine (A);Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0366] (c) substitution of an amino acid at a position corresponding to position 207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q); and / or

[0367] (d) substitution of an amino acid at a position corresponding to position 207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q).

[0368] Embodiment 11-24. The modified flavivirus NS1 polypeptide of embodiment 11-20, wherein the amino acid substitution comprises substitution of a wing domain of the NS1 polypeptide with a wing domain of a different flavivirus NS1 polypeptide.

[0369] Embodiment 11-25. The modified flavivirus NS1 polypeptide of embodiment 11-24, wherein the wing domain of a WNV NS1 polypeptide is substituted with a wing domain of ZIKV, JEV, or DENV NS 1 polypeptide.

[0370] Embodiment 11-26. The modified flavivirus NS1 polypeptide of embodiment 11-24, wherein the wing domain of a different flavivirus NS1 polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 43-46, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

[0371] Embodiment 11-27. The modified flavivirus NS1 polypeptide of any one of embodiments 11-18 to 11-26, wherein the at least one modification comprises an amino acid insertion.

[0372] Embodiment 11-28. The modified flavivirus NS1 polypeptide of embodiment 11-27, wherein the amino acid insertion comprises insertion of an ER signal sequence, a portion of a flavivirus NS protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT- independent eVLP inducing domain, a transmembrane domain, or a combination thereof.

[0373] Embodiment 11-29. The modified flavivirus NS1 polypeptide of embodiment 11-28, wherein the insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5.

[0374] Embodiment 11-30. The modified flavivirus NS1 polypeptide of embodiment 11-28 or II- 29, wherein the flavivirus nonstructural protein is NS2 or NS3.

[0375] Embodiment 11-31. The modified flavivirus NS1 polypeptide of any one of embodiments 11-28 to 11-30, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD.

[0376] Embodiment 11-32. The modified flavivirus NS1 polypeptide of embodiment II-31, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain.

[0377] Embodiment 11-33. The modified flavivirus NS1 polypeptide of embodiment 11-32, wherein the transmembrane domain comprises the amino acid sequence of the transmembraneAttorney Docket No.: VCCN-016 / 01WO 347597-2099 domain of any one of SEQ ID NOs: 20 to 34 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

[0378] Embodiment 11-34. The modified flavivirus NS1 polypeptide of any one of embodiments 11-18 to 11-33, wherein the insertion comprises an ER export sequence and wherein the export sequence comprises the YTDIEM motif (SEQ ID NO: 144).

[0379] Embodiment 11-35. The modified flavivirus NS1 polypeptide of any one of embodiments 11-18 to 11-34, wherein the insertion comprises a transmembrane domain.

[0380] Embodiment 11-36. The modified flavivirus NS1 polypeptide of embodiment 11-35, wherein the transmembrane domain is 20, 21, 22, 23, or 24 amino acids in length.

[0381] Embodiment 11-37. The modified flavivirus NS1 polypeptide of embodiment 11-36, wherein the transmembrane domain is 21 or 22 amino acids in length.

[0382] Embodiment 11-38. The modified flavivirus NS1 polypeptide of any one of embodiments 11-35 to 11-37, wherein the transmembrane domain is derived from a bacterial or viral protein.

[0383] Embodiment 11-39. The modified flavivirus NS1 polypeptide of embodiment 11-38, wherein the bacterial or viral protein is selected from the group consisting of: Epstein-Barr Virus (EBV) gp220 protein, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 M protein, Bovine viral diarrhea virus (BVDV) E2 protein, SARS-CoV-2 E protein, E. coli PmrR protein, Salmonella enterica (S. enterica) MgtU protein, Mycobacterium KdpF protein, Herpes Simplex Virus-2 (HSV-2) gH protein, Human Cytomegalovirus (HCMV) gH protein, Influenza A (FluA) M2 protein, FluA hemagglutinin protein, Measles virus hemagglutinin protein, Sendai virus (SeV) F protein, Vesicular Stomatitis Virus (VSV) G protein, Simian Vacuolating Virus 40 (SV40) Agnoprotein, Monkeypox virus (Mpox) A35R protein, and Sindbis virus (SINV) El protein.

[0384] Embodiment 11-40. The modified flavivirus NS1 polypeptide of any one of embodiments 11-35 to 11-37, wherein the transmembrane domain is derived from a platelet-derived growth factor receptor (PDGFR).

[0385] Embodiment 11-41. The modified flavivirus NS1 polypeptide of embodiment 11-40, wherein the transmembrane domain is derived from a human PDGFR (hPDGFR) or common sole PDGFR (caPDGFR)

[0386] Embodiment 11-42. The modified flavivirus NS1 polypeptide of any one of embodiments 11-35 to 11-41, wherein the transmembrane domain comprises the amino acid sequence of any one of SEQ ID NOS: 110-134, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0387] Embodiment 11-43. The modified flavivirus NS1 polypeptide of any one of embodiments 11-35 to 11-42, wherein the transmembrane domain is inserted at the N- or C-terminus of the NS1 polypeptide.

[0388] Embodiment 11-44. The modified flavivirus NS1 polypeptide of any one of embodiments 11-35 to 11-43, wherein the insertion comprises (a) a transmembrane (TM) domain and (b) an ERD or an ESCRT-independent eVLP inducing domain.

[0389] Embodiment 11-45. The modified flavivirus NS1 polypeptide of embodiment 11-44, wherein the insertions are made at the C-terminus of the NS1 polypeptide and comprise, from N-terminus to C-terminus:

[0390] (a) a TM domain and an ERD; or

[0391] (b) a TM domain and an ESCRT-independent eVLP inducing domain.

[0392] Embodiment 11-46. The modified flavivirus NS1 polypeptide of any one of embodiments 11-35 to 11-43, wherein the insertion comprises (a) a TM domain, (b) an endocytosis prevention motif (EPM), and (c) an ERD or an ESCRT-independent eVLP inducing domain.

[0393] Embodiment 11-47. The modified flavivirus NS1 polypeptide of embodiment 11-46, wherein the insertions are made at the C-terminus of the NS1 polypeptide and comprise, from N-terminus to C-terminus:

[0394] (a) a transmembrane (TM) domain, an EPM, and an ERD; or

[0395] (b) a transmembrane (TM) domain, an EPM, and an ESCRT-independent eVLP inducing domain.

[0396] Embodiment 11-48. The modified flavivirus NS1 polypeptide of any one of embodiments 11-18 to 11-47, comprising an amino acid insertion comprising an ERD, wherein the ERD comprises the sequence of any one of SEQ ID NOS: 350-387 or a sequence having at least 70% sequence identity thereto.

[0397] Embodiment 11-49. The modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-47, wherein the at least one modification is relative to a naturally occurring NS1 protein sequence from a flavivirus of the Orthoflavirvirus genus, optionally wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0398] Embodiment 11-50. The modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-49, wherein the amino acid sequence of the modified polypeptide comprises aAttorney Docket No.: VCCN-016 / 01WO 347597-2099 sequence with at least 70% identity to any one of SEQ ID NOs: 20 to 34 or a truncated sequence thereof.

[0399] Embodiment 11-51. The modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-50, wherein the amino acid sequence of the modified polypeptide comprises the sequence of any one of SEQ ID NOS: 35 to 42, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

[0400] Embodiment 11-52. The modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-51, wherein the amino acid sequence of the modified polypeptide comprises the sequence of any one of SEQ ID NOS: 278-311 and 324-336, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

[0401] Embodiment 11-53. A plurality of modified flavivirus NS1 polypeptides selected from any one of embodiments 11-17 to 11-50, wherein the plurality of NS1 polypeptides are fused into a single polypeptide chain, and wherein the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides with identical or non-identical sequences.

[0402] Embodiment 11-54. The plurality of modified flavivirus NS1 polypeptides of embodiment 11-53, wherein the at least two modified NS1 polypeptides comprise a NS1 polypeptide derived from a first flavivirus and a NSl polypeptide derived from a second flavivirus.

[0403] Embodiment 11-55. The plurality of modified flavivirus NS1 polypeptides of embodiment 11-54, wherein the first flavivirus and the second flavivirus are the same.

[0404] Embodiment 11-56. The plurality of modified flavivirus NS1 polypeptides of embodiment 11-54, wherein the first flavivirus and the second flavivirus are different.

[0405] Embodiment 11-57. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-54 to 11-56, wherein the first flavivirus and the second flavivirus are selected independently from the group consisting of: West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0406] Embodiment 11-58. The plurality of modified flavivirus NS1 polypeptides of embodiment 11-57, wherein:

[0407] (a) the first flavivirus is WNV and the second flavivirus is ZIKV;Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0408] (b) the first flavivirus is WNV and the second flavivirus is JEV;

[0409] (c) the first flavivirus is WNV and the second flavivirus is DENV;

[0410] (d) the first flavivirus is ZIKV and the second flavivirus is JEV;

[0411] (e) the first flavivirus is ZIKV and the second flavivirus is DENV; or

[0412] (f) the first flavivirus is JEV and the second flavivirus is DENV;

[0413] Embodiment 11-59. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-53 to 11-58, comprising at least three modified NS1 polypeptides with nonidentical sequences, wherein the at least three NS1 polypeptides comprise a NS1 polypeptide derived from a first flavivirus, a NS1 polypeptide derived from a second flavivirus, and a NS1 polypeptide derived from a third flavivirus.

[0414] Embodiment 11-60. The plurality of modified flavivirus NS1 polypeptides of 59, wherein the first flavivirus, the second, and the third flavivirus are selected independently from the group consisting of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

[0415] Embodiment 11-61. The plurality of modified flavivirus NS1 polypeptides of embodiment 11-60, wherein:

[0416] (a) the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is JEV;

[0417] (b) the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is DENV;

[0418] (c) the first flavivirus is WNV, the second flavivirus is JEV, and the third flavivirus is DENV; or

[0419] (d) the first flavivirus is ZIKV, the second flavivirus is JEV, and the third flavivirus is DENV.

[0420] Embodiment 11-62. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-53 to 11-61, wherein the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides, wherein the sequences of the at least two modified NS 1 polypeptides are not identical, thereby improving the breadth of immunogenicity.

[0421] Embodiment 11-63. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-53 to 11-62, comprising the amino acid sequence of any one of SEQ ID NOS:Attorney Docket No.: VCCN-016 / 01WO 347597-2099286-295, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

[0422] Embodiment 11-64. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-53 to 11-63, wherein the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice.

[0423] Embodiment 11-65. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-53 to 11-64, comprising two, three, or five modified NS1 polypeptides selected from any one of embodiments 11-17 to 11-52, wherein the sequences of the two, three, or five modified NS1 polypeptides are not identical, optionally wherein the modified NS1 polypeptides have orthogonal interfaces.

[0424] Embodiment 11-66. The plurality of modified flavivirus NS1 polypeptides of any one of embodiments 11-53 to 11-65, wherein the plurality of polypeptides is capable of assembling into a stochastic mosaic, optionally comprising six, seven, or eight modified NS1 polypeptides with identical interfaces, wherein the sequences of the six, seven, or eight modified NS1 polypeptides are not identical.

[0425] Embodiment 11-67. A modified flavivirus prM polypeptide comprising at least one modification relative to a naturally occurring prM polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

[0426] Embodiment 11-68. The modified flavivirus prM polypeptide of embodiment 11-67, wherein the characteristic is the increased ability to stabilize the conformation of naturally occurring or modified flavivirus E, improved expression, improved secretion, a decrease in immature, conformationally heterogeneous virions, or a combination thereof.

[0427] Embodiment 11-69. The modified flavivirus prM polypeptide of embodiment 11-67 or II- 68, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

[0428] Embodiment 11-70. The modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-69, wherein the at least one modification comprises an amino acid deletion.

[0429] Embodiment 11-71. The modified flavivirus prM polypeptide of embodiment 11-70, wherein the amino acid deletion comprises deletion of the pr amino acid sequence or a portion thereof, optionally wherein the pr amino acid sequence comprises the sequence of any one of SEQ ID NOs: 70-77 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0430] Embodiment 11-72. The modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-71, wherein the at least one modification comprises an amino acid substitution.

[0431] Embodiment 11-73. The modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-72, wherein the at least one modification comprises an amino acid insertion.

[0432] Embodiment 11-74. The modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-73, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 50-67 or a truncated sequence thereof.

[0433] Embodiment 11-75. The modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16, further fused directly or through a linker to a second flavivirus polypeptide.

[0434] Embodiment 11-76. The modified flavivirus E polypeptide of embodiment 11-75, wherein the second flavivirus polypeptide comprises a naturally occurring prM polypeptide or the modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-73, a naturally occurring NS 1 or the modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-52, and / or the plurality of NS1 polypeptides of any one of embodiments 11-53 to 11-63.

[0435] Embodiment 11-77. The modified flavivirus E polypeptide of embodiment 11-76, wherein the second flavivirus polypeptide is fused directly or through a linker to the N- or C-terminus of the modified flavivirus E polypeptide.

[0436] Embodiment 11-78. The modified flavivirus E polypeptide of any one of embodiments 11-75 to 11-77, wherein the second flavivirus polypeptide comprises a naturally occurring prM or the modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-73; and wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide.

[0437] Embodiment 11-79. The modified flavivirus E polypeptide of any one of embodiments 11-75 to 11-77, wherein the second flavivirus polypeptide comprises a naturally occurring NS1 or the modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-52 or the plurality of NS1 polypeptides of any one of embodiments 11-53 to 11-63; and wherein the second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

[0438] Embodiment 11-80. The modified flavivirus E polypeptide of any one of embodiments 11-75 to 11-79, further fused directly or through a linker to a third flavivirus polypeptide.

[0439] Embodiment 11-81. The modified flavivirus E polypeptide of embodiment 11-80, wherein the third flavivirus polypeptide comprises a naturally occurring prM polypeptide or the modifiedAttorney Docket No.: VCCN-016 / 01WO 347597-2099 flavivirus prM polypeptide of any one of embodiments 11-67 to 11-73, the modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-52, and / or the plurality of NS1 polypeptides of any one of embodiments 11-53 to 11-63.

[0440] Embodiment 11-82. The modified flavivirus E polypeptide of embodiment 11-81, wherein the second flavivirus polypeptide comprises a naturally occurring prM or the modified flavivirus prM polypeptide of any one of embodiments 11-67 to 11-73 and the third flavivirus polypeptide comprises a naturally occurring NS1 or the modified flavivirus NS1 polypeptide of any one of embodiments 11-17 to 11-52 or the plurality of NS1 polypeptides of any one of embodiments II- 53 to 11-63; and wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide and the third second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

[0441] Embodiment 11-83. The modified flavivirus E polypeptide of any one of embodiments 11-75 to 11-82, comprising a protease cleavage site.

[0442] Embodiment 11-84. The modified flavivirus E polypeptide of embodiment 11-79 or 11-82, comprising a protease cleavage site, wherein the protease cleavage site is located between the modified flavivirus E polypeptide and the modified flavivirus NS1 polypeptide or the plurality ofNSl polypeptides.

[0443] Embodiment 11-85. The modified flavivirus E polypeptide of embodiment 11-83 or 11-84, wherein the protease cleavage site is a naturally occurring cleavage site in a flavivirus, optionally wherein the cleavage site is a host cleavage site.

[0444] Embodiment 11-86. The modified flavivirus E polypeptide of any one of embodiments 11-75 to 11-85, wherein the modified flavivirus E polypeptide comprises the sequence of any one of SEQ ID NOS: 150-173, 176-183, 185-227, 229-270, 272-277, and 312-323 or a sequence having at least 70% sequence identity thereto.

[0445] Embodiment 11-87. A plurality of modified flavivirus E polypeptides selected from any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, wherein the plurality of polypeptides are fused into a single chimeric polypeptide chain.

[0446] Embodiment 11-88. The plurality of modified flavivirus E polypeptides of embodiment 11-87, wherein the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice.

[0447] Embodiment 11-89. The plurality of modified flavivirus E polypeptides of embodiment 11-87 or 11-88, comprising two, three, or five modified E polypeptides selected from any one ofAttorney Docket No.: VCCN-016 / 01WO 347597-2099 embodiments II- 1 to 11-16 and 11-75 to 11-86, wherein the sequences of the comprising two, three, or five modified E polypeptides are not identical, optionally wherein the modified E polypeptides have orthogonal interfaces.

[0448] Embodiment 11-90. The plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-89, wherein the plurality of polypeptides is capable of assembling into a stochastic mosaic, optionally comprising six, seven, or eight modified E polypeptides with identical interfaces, wherein the sequences of the six, seven, or eight modified E polypeptides are not identical.

[0449] Embodiment 11-91. A polynucleotide encoding the amino acid sequence of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90.

[0450] Embodiment 11-92. The polynucleotide of embodiment 11-91, wherein the polynucleotide comprises DNA.

[0451] Embodiment 11-93. The polynucleotide of embodiment 11-91, wherein the polynucleotide comprises RNA.

[0452] Embodiment 11-94. The polynucleotide of embodiment 11-93, wherein the polynucleotide comprises mRNA.

[0453] Embodiment 11-95. The polynucleotide of embodiment 11-94, wherein the mRNA comprises modified nucleosides.

[0454] Embodiment 11-96. A vector comprising the polynucleotide of any one of embodiments 11-91 to 11-95 or a plurality of polynucleotides selected from any one of embodiments 11-91 to II- 95.

[0455] Embodiment 11-97. The vector of embodiment 11-96, wherein the vector is a viral vector.

[0456] Embodiment 11-98. The vector of embodiment 11-97, wherein the viral vector is an adeno-associated virus (AAV) vector, adenovirus vector, modified vaccinia virus Ankara (MV A) vector, poxvirus vector, or Vesicular stomatitis virus (VSV) vector.

[0457] Embodiment 11-99. The vector of embodiment 11-98, wherein the adenovirus vector is Ad26, Ad34, Ad35, Ad48, or Ad5HVR48.

[0458] Embodiment II- 100. The vector of embodiment 11-96, wherein the vector is a non-viral vector.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0459] Embodiment II- 101. The vector of embodiment II- 100, wherein the non-viral vector is a plasmid.

[0460] Embodiment 11-102. The vector of embodiment II- 100, wherein the non-viral vector is a lipid nanoparticle (LNP).

[0461] Embodiment 11-103. A cell comprising the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90.

[0462] Embodiment 11-104. A cell displaying on its cell surface all or a portion thereof of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90.

[0463] Embodiment 11-105. An enveloped virus-like particle (eVLP) comprising the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90.

[0464] Embodiment 11-106. An enveloped virus-like particle (eVLP) displaying all or a portion thereof of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and II- 75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90.

[0465] Embodiment 11-107. A vaccine composition comprising the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any oneAttorney Docket No.: VCCN-016 / 01WO 347597-2099 of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, or the eVLP of embodiment 11-105 or 11-106.

[0466] Embodiment 11-108. The vaccine composition of embodiment 11-107, further comprising one or more adjuvants.

[0467] Embodiment 11-109. The vaccine composition of embodiment 11-107 or 11-108, further comprising one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0468] Embodiment II- 110. A kit comprising the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or 11-106, or the vaccine composition of any one of embodiments 11-107 to 11-109; and instructions for use.

[0469] Embodiment II- 111. A method of preventing a flavivirus infection or a symptom thereof in a subject having, or at risk of having, a flavivirus infection, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or II- 106, or the vaccine composition of any one of embodiments 11-107 to 11-109.

[0470] Embodiment II- 112. The method of preventing a flavivirus infection of embodiment II- 111, wherein the subject has, or is at risk of having, an infection of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0471] Embodiment II- 113. The method of preventing a flavivirus infection of embodiment II- 111 or II- 112, wherein the subject is a mammal or a bird, optionally wherein the subject is pregnant.

[0472] Embodiment II- 114. The method of preventing a flavivirus infection of embodiment II-113, wherein the subject is a mammal.

[0473] Embodiment II- 115. The method of preventing a flavivirus infection of embodiment II-114, wherein the subject is a human, optionally wherein the human subject is pregnant.

[0474] Embodiment II- 116. The method of preventing a flavivirus infection of embodiment II- 113, wherein the subject is a bird.

[0475] Embodiment II- 117. A method of treating a flavivirus infection or a symptom thereof in a subject having a flavivirus infection, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and II- 75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or 11-106, or the vaccine composition of any one of embodiments 11-107 to 11-109.

[0476] Embodiment II- 118. A method of inducing anti-flavivirus antibodies in a subject, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or 11-106, or the vaccine composition of any one of embodiments 11-107 to 11-109.

[0477] Embodiment II- 119. The method of embodiment II- 118, wherein the anti-flavivirus antibodies target E protein, M protein, and / or NS 1 protein, optionally wherein the anti-flavivirus antibodies target proteins of at least two flaviviruses.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0478] Embodiment 11-120. A method for inducing a flavivirus-specific immune response, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or 11-106, or the vaccine composition of any one of embodiments 11-107 to 11-109, optionally wherein the immune response provides immune protection for at least two flaviviruses.

[0479] Embodiment 11-121. The modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or II- 106, or the vaccine composition of any one of embodiments 11-107 to 11-109 for use in the treatment of a flavivirus infection or a symptom thereof.

[0480] Embodiment 11-122. Use of the modified flavivirus E polypeptide of any one of embodiments II- 1 to 11-16 and 11-75 to 11-86, the modified NS1 polypeptide of any one of embodiments 11-17 to 11-52, the plurality of modified flavivirus NS1 polypeptide of any one of embodiments 11-53 to 11-65, the modified prM polypeptide of any one of embodiments 11-67 to 11-74, or the plurality of modified flavivirus E polypeptides of any one of embodiments 11-87 to 11-90, the polynucleotide of any one of embodiments 11-91 to 11-95, the vector of any one of embodiments 11-96 to 11-102, the cell of embodiment 11-103 or 11-104, the eVLP of embodiment 11-105 or 11-106 or the vaccine composition of any one of embodiments 11-107 to 11-109 in the manufacture of a medicament for the treatment of a flavivirus infection or a symptom thereof.EXAMPLESMaterials and methodsAttorney Docket No.: VCCN-016 / 01WO 347597-2099SubcloningIndividual flavivirus sequences were ordered directly as plasmids or were subcloned to insert ordered gBlocks into vectors that were initially linearized via PCR using primers. Amino acid sequences of the resultant constructs are shown in Tables 6A-6C. Codons were optimized using Twist’s or IDT’s codon optimization tool and subsequently synthesized. The Gibson assembly cloning strategy was employed, whereby 15-base pair overlaps with the 3’ and 5’ ends of the linearized vector DNA were added to the 5’ and 3’ ends of the gene fragments, respectively. For assembly, 100 ng of genes were incubated with 50 ng of linearized vector and 2 pl of InFusion® Snap Assembly Master Mix (Takara) at 50°C for 15 minutes. The assembly mixture (2.5 pl) was used to transform 50 pl chemically competent E.coli (NEB 10 beta) and plasmid DNA subsequently isolated using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). The sequence of each plasmid was confirmed via NanoPore sequencing at Plasmidsaurus.Cell TransfectionHEK293T cells were seeded at 30,000 cells / well on a 96-well tissue culture plate (Greiner Bio- One). After approximately 6-18 hours, 100 ng of plasmid DNA was mixed with 5 pl of serum- free DMEM medium (Thermo Scientific) and 0.15 pl of BioT transfection reagent (Morganville Scientific), followed by a 5 -minute incubation at room temperature. The transfection cocktail was then added to each well containing seeded HEK293T cells. Approximately 18 hours posttransfection, the cell medium was replaced with fresh DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin. After specified times post-transfection, cell culture supernatant was collected and centrifuged at 500xg for 10 minutes to remove live cells and cell debris.Dot Blot1.5 - 2 pl of supernatant was blotted on a nitrocellulose membrane (Bio-Rad) and allowed to dry completely for 30-60 minutes. The membrane was then incubated with blocking solution (3% non-fat milk in PBST) for an hour, followed by incubation with primary antibody at a 1 : 1000 dilution (in blocking solution) for another hour. After washing with PBST three times (5 minutes with rocking each time), the membrane was incubated with an HRP-conjugated secondary antibody at 1 :5000 dilution (in blocking solution) for an additional hour. Following three more PBST washes, the membrane was incubated with Pierce™ ECL Western Blotting Substrate Mix (3ml luminol / enhancer plus stable peroxide buffer, Thermo Scientific) for 3 minutes. Dot blot images were captured using an iBright™ CL750 Imaging System (Thermo Scientific).Attorney Docket No.: VCCN-016 / 01WO 347597-2099Plasmid generation

[0481] Plasmids containing the antigen(s) of interest were codon optimized for mammalian expression and synthesized by Twist. In some cases, modifications to plasmid templates were generated by In-Fusion® Snap assembly. Here, the plasmid template was linearized via PCR and designed to contain homologous ~15-base pair overlaps with insert gene fragments synthesized by IDT. For assembly, 100 ng of insert gene fragments were incubated with 50 ng of linearized vector and 2 pl of In-Fusion® Snap Assembly Master Mix (Takara) at 50°C for 15 minutes. The assembly mixture (2.5 pl) was used to transform 50 pl chemically competent E.coli (NEB 10 beta) and plasmid DNA subsequently isolated using the ZymoPURE Plasmid Miniprep Kit (Zymo Research). The sequence of each plasmid was confirmed via NanoPore sequencing at Plasmidsaurus.Antigen expression and secretion screening by dot blot

[0482] Expi293F cells were grown to ~3 million / mL density and transfected with 1 pg / mL (plasmid DNA encoding the antigen(s) of interest using ExpiFectamine 293 Transfection Kit (Gibco) according to the manufacturer’s instructions. At 3-4 days post-transfection, cells were pelleted by centrifugation at 3,374 x g for 10 min, and clarified supernatant was collected. Cell pellets were lysed using the M-PER Mammalian Protein Extraction Reagent (Thermo Fisher) according to manufacturer’s instructions. Antigen expression and secretion were assessed by blotting 2 pl each of cell lysate and supernatant onto nitrocellulose membrane (Bio-Rad) and allowed to dry completely for 30 min. The membrane was then incubated with blocking solution (3% non-fat milk in PBST) for 30 min, followed by incubation with primary antibody diluted to 1 pg / mL in blocking solution for 1 h. After washing with PBST three times (5 minutes with rocking each time), the membrane was incubated with a HRP -conjugated secondary antibody diluted 1 :5000 in blocking solution for 1 h. Following three more PBST washes, the membrane was incubated with Pierce™ ECL Western Blotting Substrate Mix (Thermo Scientific) and imaged using an iBright™ CL750 Imaging System (Thermo Scientific). In some cases, the intensity of each dot was quantified by densitometry using Imaged (National Institutes of Health).Confirmation of antigen proteolytic cleavage

[0483] Clarified transfection supernatant was mixed at a 3 : 1 ratio by volume with 4x Laemmli sample buffer (Bio Rad), heated at 95 °C for 5 min, and loaded onto a pre-cast 4-20% SDS- PAGE gel (Bio Rad). Gels were run at 230 V for 30 min, transferred to a 0.45 pm nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio Rad), and blocked with 3% non-fatAttorney Docket No.: VCCN-016 / 01WO 347597-2099 milk in PBST (blocking buffer) for 1 h. The membrane was washed three times in blocking buffer then incubated with primary antibody diluted to 1 pg / mL in blocking solution for 1 h. The membrane was washed three times again in blocking buffer then incubated with a HRP- conjugated secondary antibody diluted 1 :5000 in blocking buffer for 1 h. Following three more PBST washes, the membrane was incubated with Pierce™ ECL Western Blotting Substrate Mix (Thermo Scientific) and imaged using an iBright™ CL750 Imaging System (Thermo Scientific).Antigen capture ELISA

[0484] Maxisorp 96-well plates (Thermo Scientific) were coated with 50 pl / well anti-E capture mAb (mouse Fc) diluted to 1 pg / mL in PBS and incubated at 4°C overnight. The next day, plates were washed 3x in 300 pl / well PBST then incubated with with 300 pl SuperBlock Blocking Buffer (Thermo Scientific) at room temperature for 30 min. Plates were washed 3x in 300 pl / well PBST, followed by addition 50 pl / well clarified transfection supernatant and incubation at room temperature with agitation (500 rpm) for 1 h. Plates were washed 3x in 300 pl / well PBST, then incubated at room temperature for 1 h with 50 pl / well anti-E primary mAb (human Fc) diluted to 1 pg / mL in SuperBlock. Plates were washed 3x in 300 pl / well PBST to remove primary antibody, followed by addition of 50 pl / well goat anti-human IgG Fc HRP- conjugated secondary antibody diluted 1 :5000 in SuperBlock and incubation at rt for 1 h. Following 6x washes in 300 pl / well PBST to remove secondary antibody, 50 pl / well of 1-Step Turbo TMB solution (Thermo Scientific) was added and incubated in the dark for 5-15 min. The reaction was stopped by addition of 50 pl / well ELISA Stop Solution (Thermo Scientific) and absorbance at 450 nm was measured using the BioTek Synergy Hl Multimode Reader (Agilent).Production and purification of eVLPs

[0485] Expi293F suspension mammalian cells (300 mL culture) were transfected with 300 total pg DNA (270 pg of construct WNV ME-137 and 30 pg of a trans-strep tag EDN plasmid, or a 9: 1 ratio) using the ExpiFectamine kit according to manufacturer’s recommendations. Cells were grown at 5% CO2 while shaking at 120 RPM in shaker flasks. Cells were harvested 6 days post transfection by centrifugation at 7,100 xg for 20 min followed by filtering supernatant through a 0.22-pm filter. Filtered supernatant was incubated with 10 mL BioLock to bind free biotin. 12 mL packed StrepTactin resin was washed with 20 mL Buffer W (100 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA) and added to supernatant. Supernatant was incubated on rotator at 4 °C overnight with StrepTactin resin. NS1 particles were purified by adding supernatant / resin slurry to a column and collecting resin. Resin was washed with 30 mL Buffer W. NS1 / Trans- strep eVLPs were eluted with Buffer BXT (20 mM Tris pH 8.0 + 150 mM NaCl + 1 mM EDTAAttorney Docket No.: VCCN-016 / 01WO 347597-2099+ 50 mM Biotin). Flow-through from StrepTactin purification was collected and incubated with 100 pL CR4354-Fab-StrepTag overnight at 4 °C on rotator. WNV ME particles were eluted by adding FT / resin slurry to a column and collecting resin. Resin was washed with 30 mL Buffer W and eVLPs were eluted with Buffer BXT. NS1 and ME particles were concentrated using a 100 kDa MWCO Amicon spin concentrator to a volume of ~0.1 mL for analysis.SDS-PAGE analysis of purified eVLPs

[0486] Denaturing SDS-PAGE analysis was performed by diluting VLPs with LDS sample buffer (containing 2-mercaptoethanol for reducing conditions) and heating samples for 5 min at 95 °C. Samples were then loaded on a 4-20% MiniPROTEAN TGX precast gel and run for 30 min at 230 V. Gels were stained using GelCode Blue staining reagent and imaged using a Thermo Fisher Scientific imager.

[0487] Dynamic light scattering of purified eVLPs

[0488] VLPs were loaded into glass capillaries (NanoTemper). Samples were then analyzed on a Nanotemper Prometheus Panta using DLS to determine the cumulant radius of the particles.TEM analysis of purified eVLPs

[0489] For transmission electron microscopy (TEM) analysis, purified eVLPs were diluted using 20 mM Tris pH 7.5, 150 mM sodium chloride. A 3.5 pL drop of diluted sample suspension was applied to TEM grid overlaid with a 3-4 nm layer of amorphous C (CF200-CU-UL, Electron Microscopy Sciences). Prior to sample application, the carbon-coated grid was plasma-cleaned for 15 seconds via a lab-made device. The 3.5 pL of sample was allowed to incubate on the carbon surface for one minute. After blotting the sample away with filter paper, each grid was twice dipped quickly (~1 second) into separate drops of de-ionized water followed by blotting with filter paper. This double washing and blotting was then repeated with two drops of 1% ammonium molybdate. For all steps, the next dipping was performed before the grid could completely dry. Finally, each grid was dipped onto a drop of 1% ammonium molybdate solution. After 15-20 seconds, the stain was blotted away with filter paper and the girds were allowed to air-dry. To collect hundreds of images rapidly, specimens were imaged on a ThermoFisher Titan Krios transmission electron microscope equipped with a Gatan Bioquantum K3 energy filter and direct electron detector. The microscope was operated at 300 kV and at liquid nitrogen temperatures, and the program SerialEM was used to collect images.Preparation of mRNA encoding antigens of interestsAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0490] Antigen designs were prepared as mRNA using in-vitro transcription from a linearized T7 plasmid vector synthesized by Genscript. Plasmid templates containing the antigen of interest were codon optimized for mammalian expression and synthesized as described above. The T7 template vector was synthesized by Genscript and contains a T7 promoter, transcription start site, 5'- and 3 '-untranslated regions (UTRs), 100 nucleotide poly(A) tail sequence, and BspQI recognition site. The antigen of interest was inserted in-frame between the 5'- and 3 '-UTRs. Plasmid sequences were confirmed by whole plasmid sequencing (Azenta). The poly(A) tail sequence was validated by Sanger sequencing (Azenta).

[0491] Vectors were linearized with BspQI-HF (New England BioLabs) at 37 °C for 2 h, and linearized vectors were purified by precipitation with sodium acetate and ethanol at -20 °C. Purified vector products were resuspended in nanopure water for in-vitro transcription (IVT) of RNA. IVT reactions were conducted using the IVTpro T7 mRNA Synthesis Kit (Takara) on a 4x reaction scale with CleanCap® Reagent AG (TriLink) and Nl-methylpseudouridine-5'- triphosphate (TriLink) in place of uridine-5'-triphosphate. Nucleoside triphosphates, IVT enzyme mix, and capping reagent were allowed to react at 37 °C for 2 h, subsequently treated with DNase I (New England BioLabs), and then purified via LiCl precipitation. Pelleted mRNA was dissolved at 1.0-4.0 pg / pL in 1 mM pH 6.4 sodium citrate buffer (Invitrogen) and stored at - 80 °C until use.Formulation ofLNPs

[0492] Lipid nanoparticles (LNPs) encapsulating mRNAs of interest were formulated using a NanoAssemblr Ignite system (Cytiva) using lipid ratios and compositions described previously in Schoenmaker et al. Int J Pharm. 2021; 601 : 120586. Briefly, ALC-0315 (Avanti Lipids), ALC-0159 (Avanti Lipids), DSPC (Avanti Lipids), and cholesterol (Sigma Aldrich) were dissolved at 25-100 mg / mL in absolute ethanol and mixed in a molar ratio of 46.3 : 1.6 : 42.7 : 9.4 to obtain the organic phase. mRNAs were prepared in 10 mM pH=4.0 sodium citrate buffer to a concentration of 0.2-0.4 mg / mL to obtain the aqueous phase. The organic phase was diluted in ethanol to target an N / P ratio of 6 after mixing, and LNPs were subsequently formulated by mixing the aqueous and organic phases using a NxGen formulation cartridge (Cytiva) at a 2 mL scale using a 12 mL / min flow rate and 3 : 1 flow ratio of the aqueous : organic phase. Following formulation, LNPs were dialyzed against 20 mM pH=7.4 tris buffer at 4 °C using 20 kDa, 2 mL Slide-A-Lyzer™ MINI Dialysis Devices (Thermo Fisher). LNPs were subsequently diluted with 50% sucrose, 20 mM tris buffer to a final concentration of 10% sucrose, concentrated using 4 mL 50 kDa Amicon® Ultra Centrifugal Filters (EMD Millipore) to a target of 0.1-0.2 mg / mLAttorney Docket No.: VCCN-016 / 01WO 347597-2099 mRNA, and sterile filtered through a 0.22 gm PES membrane. LNPs were frozen and stored at - 80 °C until use. In some cases, LNPs were purchased from Genscript using an equivalent lipid mix and formulated in PBS + 10% sucrose for storage. mRNA integrity characterization

[0493] Capillary electrophoresis was conducted on mRNA before and after packaging in LNPs. Unpackaged mRNA was diluted to 100 pg / mL, heated to 70 °C for 2 min, then cooled on ice and loaded onto the instrument. Packaged mRNA was extracted from LNPs by mixing 5 pL of LNP with 10 pL of LNP digestion buffer (30% v / v ethanol + 20% v / v Triton X-100) and heating the resultant solution to 37 °C for 20 min, then to 70 °C for 2 min. Samples were cooled on ice and loaded onto the instrument. RNA integrity and purity was validated using capillary electrophoresis on a Fragment Analyzer 5200 System (Agilent) using the DNF-471 15nt RNA Kit (Agilent) according to the manufacturer’s protocol. RNA integrity was determined via smear analysis using the instrument software.Dynamic Light Scattering of LNPs

[0494] LNPs were diluted 1 : 10 in phosphate-buffered saline and subsequently loaded into glass capillaries. Samples were then analyzed on a Prometheus Panta (Nanotemper) using dynamic light scattering to determine the cumulant diameter and poly dispersity index of the particles. mRNA concentration in LNPs

[0495] mRNA concentration and loading efficiency were determined using the Quant-it™ RiboGreen RNA Assay Kit (Thermo Fisher). LNPs and rRNA standards were diluted in 10 mM tris + 1 mM EDTA pH=8.0 (TE) buffer, and 50 pL of each diluted sample or rRNA standard was loaded into a black 96 well plate. Samples were then further diluted with 50 pL of TE buffer to determine unencapsulated RNA concentration or TE buffer + 2% Triton X-100 to determine total RNA concentration (each in duplicate). Samples were heated at 37 °C for 5 min to digest LNPs, then cooled to room temperature and treated with 100 pL of diluted RiboGreen reagent (1 : 1000 in TE buffer). Samples were incubated for 5 min in the dark and subsequently read using a Biotek Synergy Hl plate reader at excitation / emission wavelengths of 485 / 528 nm. The rRNA standards were fit to a sigmoidal 4 parameter logistic curve in GraphPad Prism 9 to determine the concentration of mRNA in samples.Evaluation of in vitro potency of RNA and LNPs

[0496] Cell -based assays were used to validate protein expression and relative potency of mRNA encoded antigen constructs. Expi293F cells at a density of 1 x 106cells / well in a 24 wellAttorney Docket No.: VCCN-016 / 01WO 347597-2099 plate were treated with 1 pg / mL mRNA complexed with Messenger Max (Thermo Fisher) in Opti-MEM media (Gibco) according to the manufacturer’s protocol. After 20-24 h, an aliquot of cells and supernatant were transferred to a 96w plate for analysis of cell surface NS1 expression via flow cytometry and the remaining cells and supernatant were processed as above for dotblot analysis.

[0497] To evaluate cell-surface antigen expression, cells were spun down at 500 x g for 5 min and supernatant was discarded. After washing with 200 pL / well PBS, cells were treated with 200 pL of 1 :1000-2000 Live-or-dye 665 / 685 (Biotium) or Live-or-dye 510 / 550 diluted in PBS for 20 min at room temperature in the dark. Cells were then spun down at 500 x g for 5 min, media was removed, and cells were treated with 50 pL diluted primary antibody (0.5-5.0 pg / mL) in flow buffer (PBS + 1% heat inactivated FBS + 1 mM EDTA) at 4 °C for 30-60 min. Primary antibodies were prepared by GenScript with mouse or human Fc domains and stored in pH=7.4 TBS. Cells were then spun down at 500 x g for 5 min, media was removed, and cells were treated with 50 pL 1 :500 diluted secondary antibody in flow buffer at 4 °C for 30-60 min. Secondary antibodies were goat anti-mouse or goat anti-human IgG conjugated with R- phycoerythrin (Southern Biotech) or Alexa Fluor 405 (Thermo Fisher). Cells were then spun down at 500 x g for 5 min, media was removed, and cells were washed twice with 200 pL flow buffer. Finally, cells were resuspended in 100 pL flow buffer and analyzed using a Cytek Aurora flow cytometer. The media fluorescence intensity of PE and / or Alexa Fluor 405was determined using FlowJo, and data were plotted in GraphPad Prism 9.Vaccine formulation and administration

[0498] Female BALB / c mice (age 7-8 weeks) were purchased from Charles River Laboratories and acclimated for at least one week in a contract vivarium (Labcorp) prior to immunization. LNPs were formulated in 20 mM tris, 10% sucrose, pH=7.4 to deliver 2 pg in 100 pL volume. Mice were injected intramuscularly with 2 x 50 pL doses of antigen in each hind leg, to deliver 100 pL of vaccine formulation per immunization. To obtain blood via retroorbital bleeding, a capillary tube was inserted into the medial canthus. Blood was then transferred to a microtainer blood collection tube (BD 365967), allowed to clot at room temperature for approximately 2 hours, and then spun down at 4000 rpm in a tabletop centrifuge. The serum layer was transferred to an Eppendorf tube and frozen at -80 °C. Prior to subsequent assays, serum was heat inactivated at 56 °C for 30 min. All mouse studies were conducted in accordance with IACUC- approved protocols.Generation ofLuminex BeadsAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0499] Carboxylated MagPlex microspheres (Luminex Corporation) were used for antigen conjugation. A distinct region of beads was assigned to each target analyte protein. The carboxyl surfaces of the beads were activated with ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to facilitate attachment of the target analyte proteins or bovine serum albumin off-target protein (Fisher Scientific) via reaction with the lysine side chains of the proteins.Luminex Assay

[0500] Serum samples were serially diluted 50-fold in Luminex diluent (1% non-fat milk, 5% fetal bovine serum, 0.05% Tween-20 in phosphate buffered saline, pH 7.4). 50 pL of diluted serum was added to 25 pL of antigen-conjugated microsphere suspension (at least 1000 microspheres / antigen). After a 1-hour incubation, microspheres were washed three times with Luminex wash buffer (0.1% fetal bovine serum and 0.02% Tween-20 in phosphate buffered saline, pH 7.4). Microspheres were then incubated with in 100 pL of goat anti-mouse IgG R- phycoerythrin (2 pg / mL final concentration, SouthemB iotech #1030-09) for 30 minutes. Results were read on a Luminex xMAP Intelliflex system, and the median fluorescence intensities of the bead region corresponding to each analyte were used for data analysis.Production of single-round infectious reporter virus particles for neutralization and ADE assays

[0501] Standard (‘ Std’) reporter virus particles of DENV2, ZIKV, and WNV were produced by co-transfection of HEK-293T / 17 cells with (i) a plasmid expressing a WNV subgenomic replicon encoding GFP in place of structural genes (as detailed in Pierson et al. Virology. 2006; 346: 53-65) and (ii) a plasmid encoding C-prM-E structural genes from the following viruses: DENV2 16681 (as detailed in Ansarah-Sobrinhoet al. Virology. 2008; 381 : 67-74), WNV NY99 (as detailed in Pierson et al. Virology. 2006; 346: 53-65), and ZIKV H / PF / 2013 (as detailed in Dowd al. Cell Rep. 2016; 16: 1485-1491). These plasmids were kindly provided by the National Institute of Allergy and Infectious Diseases (NIAID). Briefly, 8 x 10A5 HEK-293T / 17 cells were plated in each well of a 6-well plate, The following day each well was co-transfected with 1 pg of replicon-encoding plasmid and 3 pg of C-prM-E-encoding plasmid using Lipofectamine 3000 (Cat# L3000-015; ThermoFisher Scientific) according to the manufacturer’s instructions. Four hours post-transfection, media was replaced with low-glucose DMEM (ThermoFisher Scientific) containing 7% FBS and 100 U / mL penicillin-streptomycin (i.e. low-glucose DMEM complete) and cells were transferred to 30°C in 5% CO2. Virus-containing supernatant was harvested at days 3-4 post-transfection, passed through a 0.22 pm Steriflip filter (Millipore-Sigma), pooled,Attorney Docket No.: VCCN-016 / 01WO 347597-2099 aliquoted, and stored at -80°C. Mature (Mat) preparations of reporter virus particles with increased efficiency of prM cleavage were produced as above by co-transfecting plasmids encoding the replicon, structural genes, and human furin (synthesized by Twist) at a 1 :3: 1 mass ratio.

[0502] Infectious titers of reporter viruses were determined by infection of Raji cells stably expressing DCSIGNR (Raji-DCSIGNR) cells, using 2-fold serial dilutions of virus stocks. At 2 days post-infection, cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences), and %GFP positive cells quantified by flow cytometry (Cytek Aurora). Raji-DCSIGNR cells were kindly provided by the NIAID and are described in detail in Davis et al. J Virol. 2006; 80(3): 1290-301.Neutralization and antibody-dependent enhancement (ADE) of infection assays

[0503] Stocks of reporter virus particles diluted to 5-10% final infectivity were incubated with 5-fold serial dilutions of heat-inactivated serum (56°C for 30 min) or purified monoclonal antibodies for 1 hr at 37°C before addition of 2e5 Raji-DCSIGNR cells (neutralization assays) or K562 cells. After incubation for 2 days at 37°C, cells were fixed in 2% paraformaldehyde and GFP positive cells were quantified either by flow cytometry (Cytek Aurora) or high-content imaging (Molecular Devices ImageXpress). Infection was normalized to conditions without antibody and analyzed using a 4-parameter logistic regression model with interpolation of serum dilution (NT50) that results in 50% inhibition of infection (GraphPad Prism).Example 1 : Designing modified E proteins and NS1 proteins

[0504] As depicted in FIG. 1, traditional pr / M-E-based flavivirus eVLPs bud into the ER lumen and transit through the trans-Golgi network, where particles undergo a maturation process characterized by pr cleavage by cellular furin, resulting in secreted particles of varying degrees of maturity. Therefore, E proteins are modified through targeted engineering to enable mRNA- driven pr-free eVLP display of fully mature forms, selectively exposing epitopes of neutralizing or broadly neutralizing antibodies. Among other methods, direct or indirect incorporation of ESCRT-recruiting domains (ERDs) into E protein may enable a pr-free eVLP platform to drive the production of homogeneous, fully mature particles (FIG. 2). E protein assembly can happen either in the ER or cell surface, driven by ESCRT recruitment. The lack of pr results in secreted particles that are fully mature. Modified E proteins are screened for incorporation of naturally occurring or modified M protein, insertion of ERD, insertion of transmembrane domains from NS proteins, and mutations that ablate immunodominant ADE-inducing epitopes within the E fusion loop (FIG. 3).Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0505] As depicted in FIG. 4, NS1 is engineered for mRNA-delivered chimeric and mosaic oligomers displaying epitopes that induce protective cross-flavivirus-reactive antibodies while eliminating regions involved in pathogenesis.

[0506] As shown in FIG. 5, optimized ERDs are determined through assessing affinity and cellular localization of ESCRT recruitment. Multiple ERD sequences are also screened for their ability to produce eVLPs.

[0507] To test the properties of various construct designs, such as ability to induce secretion of eVLPs, 293T cells were transfected with plasmids encoding either prME or ME of different flaviviruses and additionally co-transfected with plasmids encoding ERD components.Assessment of E protein expression

[0508] 293T cells were transfected with plasmids encoding either prME or ME of Zika virus (ZIKV), Japanese encephalitis virus (JEV), or yellow fever virus (YFV). As shown in FIG. 9, E protein expression was assessed in cell supernatant (FIG. 9A, top and bottom; and FIG. 9B, top) or in cell lysate (FIG. 9B, bottom). The results indicate that prME-derived eVLPs are secreted from the cells, but those derived from ME only are sequestered in the cell and only liberated following cell lysis.

[0509] 293T cells were transfected with plasmids encoding either prME or ME of ZIKV or YFV. Plasmids encoding prME or ME were co-transfected with those encoding ERD components (YFV NS3, ZIKV NS1-3, or MERS-1227 EABR). E protein expression was assessed in cell supernatant (S) or from cell lysate (C) using either a rabbit polyclonal anti-ZIKV E (FIG. 10A) or with a mouse monoclonal pan-flavivirus E antibody (FIG. 10B). The results suggest that NS3 increased secretion efficiency of prME-, but not ME-derived eVLPs.

[0510] 293T cells were transfected with plasmids encoding components as shown in Table 8. E protein expression was assessed in cell lysate (FIG. 11 A) and cell supernatant (FIG. 11B). The results indicate that prME-derived eVLPs are secreted from the cells, but those derived from ME only are sequestered in the cell and only liberated following cell lysis.Table 8: Components of plasmids used to transfect 293 T cellsAttorney Docket No.: VCCN-016 / 01WO 347597-2099Example 2: Antigen design

[0511] FIGs. 6A-6D depict four types of E antigen designs, which are also applicable for NS 1. The four types of antigen designs are eVLPs displaying E proteins as (1) a chimeric antigen (FIG. 6A), (2) a controlled mosaic (FIG. 6B), (3) a stochastic mosaic (FIG. 6C), or (4) fully orthogonal mixtures (FIG. 6D) capable of being delivered as a single mRNA vaccine formulation. Chimeric E proteins are produced by combining sequences from many E proteins all into a single sequence capable of producing mature eVLPs. Controlled mosaics leverage the viral icosahedral surface symmetry to control the mosaic formation to have either 2, 3, or 5 components (based on the 2-fold, 3-fold, or 5-fold symmetries of E proteins on the virion surface). Stochastic mosaics are designed to randomly associate similar E protein sequences, or at least those that have complementary interfaces. Finally, orthogonal eVLPs can be produced by ensuring only specific compositions of modified E proteins are able to associate with one another.Example 3: M-E-NS1 polypeptide design enables display of E and NS1 on separate eVLPs

[0512] Flavivirus M (without pr), E, and NS1 proteins were encoded as a single polypeptide, preceded by a signal sequence from Japanese encephalitis virus (JEV) capsid protein. In thisAttorney Docket No.: VCCN-016 / 01WO 347597-2099 configuration, the native protease cleavage sites between M, E, and NS1 are present, allowing proper cleavage of individual proteins and self-assembly of ME into eVLPs.

[0513] To facilitate ESCRT-driven nanoparticle (EDN) display of NS1 on separate eVLPs, the C-terminus of NS1 was fused to an artificial transmembrane (TM) domain, followed by an endocytosis prevention motif (EPM), and an EDN sequence tag, as depicted in FIG. 12. Table 6B provides a panel of constructs, with TM domains and EDN tag sequences as indicated in the description, that were screened in the context of M-E-NS1 design for WNV and ZIKV. Table 6C provides a list of down-selected antigen designs that were synthesized as mRNA for mouse immunization studies. In some cases, as exemplified by construct FLV.6004 in Table 6C, instead of inserting EPM and EDN tag downstream of NS 1 to facilitate budding of membranebound NS1, a TM domain from platelet-derived growth factor receptor (PDGFR) was introduced, which promotes exosome budding in an ESCRT-independent fashion.Expression and secretion of WNV E and NS1 from M-E-NS1 polypeptide designs

[0514] FIG. 13A provides a schematic of constructs representative of the design presented in FIG. 12, with asterisks indicating previously established NS 1 KO mutations. Expi293F cells were transfected with plasmids encoding the constructs shown in Table 9A below.Table 9A: Constructs corresponding to dot blot of FIGS. 13B-13C

[0515] In Table 9A, EPM denotes endocytosis prevention motif; hPDGFR denotes human PDGFR; csPDGFR denotes common sole PDGFR; and caPDGFR denotes Chinese alligator PDGFR.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0516] Four days post-transfection, cell lysate and supernatant from transfected cells were analyzed by dot blot for expression and secretion of E proteins (FIG. 13B) and NS1 proteins (FIG. 13C) using mAb 4G2 and 22NS1, respectively. The dot blot layout is provided in Table 9B below.Table 9B: Dot blot layout for FIGS. 13B-13C.Expression and secretion of ZIKV E and NS1 from M-E-NS1 designs

[0517] FIG. 14A provides a schematic of additional construct designs, with bars within M and E segments representing native transmembrane (TM) domains. Expi293F cells were transfected with plasmids encoding the constructs shown in Table 10A below.Table 10A: Constructs corresponding to dot blot of FIGS. 14B-14CAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0518] Four days post-transfection, cell lysate and supernatant from transfected cells were analyzed by dot blot for expression and secretion of E proteins (FIG. 14B) and NS1 proteins (FIG. 14C) using mAb ZV-67 or 1G65.3, respectively. The dot blot layout is provided in Table 9B below.Table 10B: Dot blot layout for FIGS. 14B-14C.

[0519] As shown in FIGS. 13B-13C and FIGS. 14B-14C, the M-E-NS1 polypeptide design was compatible with expression and secretion of E and NS1 proteins for both WNV and ZIKV.Attorney Docket No.: VCCN-016 / 01WO 347597-2099Additionally, the cell lysate and supernatant of Expi293F cells transfected with the constructs depicted in FIG. 15A, in addition to WT WNV prME and WT WNV ME controls were analyzed by non-reducing SDS-PAGE and western blotting using anti-E mAb 4G2 and anti-NSl mAb 22NS1. As shown in FIGS. 15B-15C, secreted E proteins were properly cleaved from the M-E-NS1 polypeptide.

[0520] Secreted WNV E proteins are particle-associated

[0521] Expi293F cells were transfected with the constructs described in Table 11A below, in addition to control plasmids (plasmids encoding WT WNV prME and ME, and soluble NS1 (WNV NS1-007) or constructs described in Table 11B below, in addition to control plasmids (plasmids encoding WT ZIKV prME and ME).Table 11 A: Constructs corresponding to FIG. 16 transfected in Expi293F cellsTable 1 IB: Constructs corresponding to FIG. 17 transfected in Expi293F cellsAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0522] Four days post-transfection, clarified supernatant from cells transfected with WNV constructs (Table 11 A) was analyzed by sandwich ELISA using anti-E mAb E16 with human Fc (hE16) for antigen capture and anti-E mAb E16 with mouse Fc (mE16) for detection. Purified soluble monomeric E protein (sE) was included as a control for the ELISA.

[0523] Supernatant from cells transfected with ZIKV constructs (Table 1 IB) was analyzed by sandwich ELISA using anti-E mAb EDE1-C10 with human Fc (hEDEl-ClO) for antigen capture and anti-E mAb EDE1-C10 with mouse Fc (mEDEl-ClO) for detection. As shown in FIG. 16 and FIG. 17, the sandwich ELISAs confirmed eVLP display of secreted E proteins.

[0524] Lastly, eVLPs generated via transfection of Expi293F cells with plasmids encoding the WNV ME-137 construct and trans-strep tag were purified according to the schematic of FIG. 18. Biochemical and biophysical characterization of purified eVLPs was performed via western blot, Coomassie stain, and dynamic light scattering (DLS) analysis of ME and NSl eVLPs. ME and NS1 eVLPs were eluted with or without addition of CR4354, a mAb that neutralizes WNV infection. Western blot and Coomassie stain analysis of ME and NS1 eVLPs were conducted under non-reducing (-BME) and reducing (+BME) conditions.

[0525] Dynamic light scattering analysis shows presumed particles of ~50 nm in both the supe and the purified samples. The primary peak in the pull-down samples is slightly smaller and less broad than signal observed in the supernatant.

[0526] As shown in FIG. 19A, elution of E protein (indicated by signal from anti-E mAb E16) required use of CR4354 for pull-down, as expected. No signal from E16 was seen in the absenceAttorney Docket No.: VCCN-016 / 01WO 347597-2099 of CR4354 pull-down, indicating that purification steps are specific. Elution of NS1 protein was seen both with and without CR4354, indicating that the initial StrepTactin (ST) purification did not fully deplete NS 1 / Trans-Strep particles (NS1 particles contain StrepTactin). As a result, additional particles were eluted when the CR4354-Fab pulldown is conducted. 22NS1 signal for detecting NS1 proteins was lost under reducing conditions, indicating that mAb 22NS1 only recognizes the epitope on NS1 under non -reducing conditions.

[0527] Coomassie stain (FIG. 19B) also confirmed purified eVLPs displayed E or NS1 proteins from the M-E-NS1 design. As shown in FIG. 19C, DLS analysis demonstrated presumed particles of about 50 nm in both the supernatant (supe) and the purified samples (ST pull-down and CR4345 pull-down), with the primary peak in the pull-down samples being slightly smaller and less broad than the signal observed in the supernatant.

[0528] Structural analysis of the purified eVLPs from the M-E-NS1 design via TEM, as shown in FIG. 20, further demonstrated that E and NS1 proteins are displayed on separate eVLPs.Example 4: M-E-NS1 design elicits neutralizing antibodies with superior quality than traditional prM-E design

[0529] WNV M-E-NS1 designs (M is without pr) fused to three different TM domains (common sole fish PDGFR (csPDGFR), measles HA, or EBV gp220) with or without EPM and EABR (FLV.6001-FLV.6006 in Table 6C), along with WNV prME and ME designs, were evaluated in mouse immunization studies. To mitigate potential toxin-like activity of NS1, the NS1 component of these vaccine antigens (WNVI NY99 NS1; SEQ ID NO: 38) encoded 4 mutations previously shown to abrogate cellular binding (W115 A, W118 A, G119A relative to SEQ ID NO: 26) and internalization (N207Q relative to SEQ ID NO: 26). Serum was collected from each mouse on day 21 and day 35 post immunization. Serial dilutions of serum from individual mice immunized with the vaccine constructs were incubated with a standard preparation of WNV expressing a GFP reporter prior to addition to Raji cells stably expressing DC-SIGNR, a flavivirus attachment factor. Infected cells were enumerated using a high-content imager (Molecular Devices ImageXpress). The serum dilution factor of serum collected on day 21 and day 35 post immunization that results in 50% inhibition of infection (NT50) was determined for each of the 5 mice per group.

[0530] As shown in FIG. 21, WNV M-E-NS1 fused to TM domain from csPDGFR without EPM and EDN tag (construct FLV.6004) elicited neutralizing antibodies after prime (day 21) or boost (day 35). This initial evaluation of neutralizing antibody responses was performed using a standard (Std) preparation of WNV, which contains incompletely mature particles that retainAttorney Docket No.: VCCN-016 / 01WO 347597-2099 uncleaved pr. The presence of pr on Std virus preparations increases the exposure of undesirable epitopes such as the fusion loop on E protein targeted by antibodies with a high potential to mediate antibody-dependent enhancement (ADE) of infection as they incompletely neutralize infectivity even at high concentrations.

[0531] Neutralization of standard (Std) and mature (Mat) preparations of WNV was then demonstrated using mAb El 6, which targets a highly exposed epitope in E domain III, and mAb E53, which targets the cryptic E domain II fusion loop epitope that is preferentially exposed on incompletely mature particles, as shown in FIG. 22A. Neutralizing antibody titers against structurally mature (Mat) virions that lack pr more accurately predict protection from flavivirus infection than those against standard virus, highlighting the importance of assessing neutralizing antibody (nAb) quality.

[0532] Neutralization of standard (Std) and mature (Mat) preparations of WNV by mouse immune sera, elicited by immunization with the construct designs, was then evaluated and mean NT50 per mouse obtained from three independent experiments was calculated. P-value (two- tailed) was obtained from a paired t-test. As shown in FIG. 22B, antibodies elicited by the WNV prM-E construct (FL V.6009) neutralized Std WNV more potently than Mat WNV, and neutralizing Ab responses elicited by pr-free designs FLV.6004 and FLV.6010 (WNV M-E-NS1 and WNV M-E, respectively) were not sensitive to virion maturation state, which is a feature of protective responses. Overall nAb titers elicited by FLV.6004 was higher than that elicited by FLV.6010, suggesting an additional benefit for including NS1 in M-E antigen design.Antibodies elicited by pr-free WNV ME antigens have diminished potential to enhance infection of autologous and heterologous flaviviruses

[0533] To further evaluate the quality of elicited antibody responses, the ability of immune sera to enhance infection of both autologous (WNV) and heterologous (ZIKV, DENV2) flaviviruses was measured. Serial dilutions of pooled sera collected at day 49 from mice immunized with the constructs in Table 12 below were incubated with a standard preparation of WNV (FIG. 23 A), ZIKV (FIG. 23B), or DENV2 (FIG. 23C) expressing a GFP reporter prior to addition to K562 cells, which express FcgRIIa and are poorly infected in the absence of IgG antibodies. Infected cells were enumerated by flow cytometry.Table 12: Constructs corresponding to FIGS. 23A-23C and FIGS. 24A-24BAttorney Docket No.: VCCN-016 / 01WO 347597-2099

[0534] As shown in FIG. 23A-23C, sera from mice immunized with pr-free WNV M-E and WNV M-E-NS1 constructs enhanced autologous and heterologous infection to a lower magnitude compared to sera from mice immunized with WNV prM-E constructs. These results suggest that pr-free WNV M-E and WNV M-E-NS1 constructs elicited a lower prevalence of cross-reactive, poorly neutralizing antibodies.

[0535] Serial dilutions of pooled sera collected at day 35 from mice immunized with the constructs of Table 12 were then assessed for binding to soluble E (sE) proteins of WNV or ZIKV in a Luminex assay. As shown in FIG. 24, the diminished potential of pr-free constructs to mediate antibody-dependent enhancement (ADE) of infection was not explained by overall lower levels of antibodies against E protein.

[0536] Together, these results suggest a higher quality of antibody responses was elicited by pr- free antigens, as indicated by insensitivity of neutralizing activity to virion maturation state and diminished capacity to mediate ADE of infection.Example 5: A longer JEV signal sequence elicits higher neutralizing antibody titers than a shorter signal sequence

[0537] Next, the impact of signal sequence length on the immunogenicity of WNV M-E constructs was studied. As shown in FIG. 25, FLV.6013, which includes a longer version of the JEV signal sequence (JEV ss) upstream of M, elicited higher neutralizing antibody titers compared to FLV.6010, which has a shorter version of the JEV ss. Thus, the length and possibly identity of signal sequences impact immunogenicity.Example 6: Screening of additional membrane-bound NS1 antigen designs for expression and secretion

[0538] To develop NS1 vaccine antigens that can be delivered independently of ME-based antigens, additional membrane-bound NS1 antigens fused to EDN tag were designed to enable dual display on the cell surface and on secreted eVLPs. To elicit broad antibody responses against NS1 proteins from different flaviviruses, a series of NS1 homodimers (from the same flavivirus), heterodimers and heterotrimers (both from different flaviviruses) were encoded as a single chain separated by a short linker sequence.

[0539] The NS1 wing domain has been reported to engage target cells, and several wing domain-targeting antibodies have been found to be protective in vivo (see, for example, Chew etAttorney Docket No.: VCCN-016 / 01WO 347597-2099 al. Antiviral Res. 2024; 227: 105915). Therefore, mosaic NS1 constructs (such as those having a sequence set forth in any one of SEQ ID NOS: 40, 41, and 43) were designed by swapping in the wing domain from a heterologous flavivirus NS1.

[0540] As described in Example 4 above, 4 mutations previously shown to abrogate NS1 cellular binding (W115 A, W118 A, G119 A) and internalization (N207Q) were introduced into all designs to avoid eliciting toxin-like activity via immunization. Construct sequences for the designs are listed in Table 6B and design strategies are depicted in FIGS. 26A-26E as follows:

[0541] FIG. 26A: NS1 homodimers were designed by linking two identical copies of NS1 from WNV, DENV1, JEV, or ZIKV. These NS1 homodimer designs encode N-terminal purification tag (strep tag II), followed by SGSG linker and the two copies of WNV NS1 that are also separated by SGSG linker. The C-terminus of the second copy of NS1 is fused to the EBV gp220 TM domain and MPMV.HIV EDN tag.

[0542] FIG. 26B: Mosaic design encoding 2 copies of NS1. The wing domain in the second copy of NS1 was swapped out with NS1 from a different flavivirus to form a mosaic NS1 construct.

[0543] FIG. 26C: Heterodimeric designs encode two copies ofNSl, each from a different flavivirus. Mismatched NS1 proteins were adopted in the first and second copies ofNSl protein, forming NS 1 heterodimers.

[0544] FIG. 26D: Heterotrimeric designs encode three copies of NS1, each from a different flavivirus. Specifically, NS1 heterotrimers were designed by selecting the NS 1 protein from WNV, DENV1, JEV, and ZIKV.

[0545] FIG. 26E: Transmembrane domains from 16 different proteins screened in the context of the homodimeric WNV NS1 base construct. The transmembrane (TM) domain (adopted from Epstein-Barr Virus (EBV) gp220 antigen) in WNV NSl_004-2 (SEQ ID NO: 278) was replaced with TM domains from 16 viral or bacterial proteins including SARS-CoV-2 M protein, Bovine viral diarrhea virus (BVDV) E2 protein, SARS-CoV-2 E protein, E. coli PmrR protein, Salmonella enterica (S. enterica) MgtU protein, Mycobacterium KdpF protein, Herpes Simplex Virus-2 (HSV-2) gH protein, Human Cytomegalovirus (HCMV) gH protein, Influenza A (FluA) M2 protein, Influenza A hemagglutinin protein, Measles virus hemagglutinin protein, Sendai virus (SeV) F protein, Vesicular Stomatitis Virus (VSV) G protein, Simian Vacuolating Virus 40 (SV40) Agnoprotein, Monkeypox virus (Mpox) A35R protein, and Sindbis virus (SINV) El protein. The sequences of these constructs where the TM domain was replaced are provided as SEQ ID NOS: 296-311 and further described in Table 6B. To assess the effect solely from theAttorney Docket No.: VCCN-016 / 01WO 347597-2099TM domain, only the TM domain in the WNV NSl_004-2 construct was replaced, the EBV gp220 cytoplasmic tail was retained in the TM screening constructs.

[0546] In order to recognize NS1 antigens from different flaviviruses, two cross-reactive monoclonal antibodies 1G5.3 and 2B7 were used for detection. However, the degree of reactivity to NS1 of WNV, DENV1, JEV, and ZIKV was unclear. Therefore, a dot blot assay was performed using NS 1 proteins of WNV, DENV1, JEV, and two different strains of ZIKV (Uganda and Suriname) to determine the degree of reactivity. As shown in FIG. 27, 1G5.3 mAb (left panel) reacted strongest to both strains of ZIKV, followed by WNV and then JEV, and had no reactivity to DENV1. 2B7 mAb reacted strongest to DENV1, moderately to ZIKV strains, weakly to WNV, and had no reactivity to JEV.

[0547] To measure the amount of membrane-bound NS1 protein in the supernatant of transfected HEK293T cells, a dot blot was performed using a cross-reactive antibody 1G5.3 as the detection antibody. Unexpectedly, despite the success of expressing WNV NS 1 homodimer on eVLPs, homodimers of ZIKV and JEV NS1 protein showed poor expression in eVLPs, as seen in FIGS. 28A-28B

[0548] As shown in FIG. 28A-28B, the addition of a long SG linker between strep tag II and the first copy of NS1 also severely compromised the expression of membrane-bound WNV NS1 homodimer (WNV_004_2 in FIG. 28A-28B). Similarly, mosaic and heterotrimer designs also demonstrated poor membrane-bound expression. Flavivirus NS1 heterodimers such as WNV- ZIKV and WNV-JEV homodimers, surprisingly, induced a similar or higher level of eVLP when compared to WNV homodimer (WNV_004 in FIG. 28A-FIG.28B).

[0549] Additionally, since 1G5.3 mAb does not react to DENV1 NS1, as shown in FIG. 27, a separate dot blot was carried out using 2B7 mAb. As shown in FIG. 29, heterodimers like WNV-DENV1 and ZIKV-DNEV1 showed strong expression.Transmembrane domain screening for WNV NS1 homodimer

[0550] It is known that the transmembrane (TM) domain not only impacts the expression of antigens anchored to it but also contains a sorting signal that directs the protein to its destination. Both of these functions can significantly affect the efficiency of EDN-driven eVLP budding. Therefore, in addition to the EBV gp220 TM domain used in WNV NSl_004, TM domains from 16 viral and bacterial proteins were selected, and their impact on eVLP formation was assessed by dot blot assay using 22NS1 mAb, as shown in FIG. 30A. Flow cytometry and cell ELISA were performed to measure the cell surface and overall antigen expression, respectively. The effect of antigen expression on EDN activity is demonstrated by plotting the cell surfaceAttorney Docket No.: VCCN-016 / 01WO 347597-2099 expression (FIG. 30B) or total cell expression (FIG. 30C) level on the x-axis and EDN activity on y-axis. Overall, among the screened TM domains, bacterial and viral TM domains showed a comparable result, with EBV gp220, HCMV gH, M. tuberculosis Kdpf, and Measles HA being the top performers in EDN activity. As shown in FIG. 30C, EDN activity appears to positively correlate with total cell expression of NS1, with the exception of Measles HA. Compared to candidates with a similar level of antigen expression, such as flu A M2 and WNV NS2a, NS1 anchored to the TM domain of Measles HA seemed to induce a much higher level of eVLP.

[0551] Furthermore, the length of TM domain has been reported to be an important factor that determine the cellular destination of a given protein, which may significantly affect eVLP formation. Therefore, EDN activity data was plotted against the length of the TM domain, as predicted by DeepTMHMM. DeepTMHMM is a deep learning protein language model-based algorithm which is described in detail in Hallgren et al. bioRxiv. 2022.04.08.487609. As shown in the FIG. 31, TM domains shorter than 20 or longer than 24 amino acids appeared to result in poorer EDN activity, and TM domains that are 21 or 22 amino acids in length appeared to be preferable for inducing eVLP.Example 7: Monomeric, homodimeric, and heterodimeric NS 1 antigens are immunogenic

[0552] NS1 antigen designs provided as FLV.6015-FLV.6029 in Table 6C and further detailed in Table 13 below were selected and synthesized as mRNA for mouse immunization studies, as depicted in FIG. 32.Table 13: Constructs corresponding to FIGS. 32-35Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0553] These designs included monomeric (WNV), homodimeric (WNV), and heterodimeric (WNV / JEV and ZIKV / DENV1) NS1 antigens with or without an EDN tag. For monomeric WNV NS1, membrane-anchored (+ / - EDN) and soluble constructs (no TM domain) were included. For the latter, constructs with either wildtype NS1 or NS1 encoding 4 mutations previously shown to abrogate cellular binding (W115 A, W118 A, G119 A) and internalization (N207Q), as described in Example 4 above, were evaluated.Attorney Docket No.: VCCN-016 / 01WO 347597-2099

[0554] The mRNA constructs of Table 13 were used to transfect Expi293F cells. In vitro potency assays were performed to determine levels of cell surface expression, which was measured by flow cytometry using anti -NS 1 mAb 1G5.3 (FIG. 33) or anti -NS 1 mAb 2B7 (FIG. 34) and calculated as % geometric mean fluorescence intensity (% gMFI) relative to mock- transfected cells. In vitro potency assays were also performed to determine levels of total cell- associated and secreted NS1 protein (FIG. 35). As shown in FIG. 35, NS1 constructs with an EDN tag (for example, FLV.6018 and FLV.6025) resulted in more efficient secretion than corresponding constructs without an EDN tag (for example, FL V.6022 and FLV.6027).

[0555] Additionally, immunogenicity of the NS1 constructs was assessed by measuring the potency and breadth of binding antibodies in pooled mouse sera (sera collected at day 35 from the five mice in each immunization group were pooled at a 1 : 1 : 1 : 1 : 1 ratio) against a panel of soluble flavivirus NS1 proteins conjugated to beads in a Luminex platform. Sera were assayed at dilutions of 1 :30-1 :2343750 using a 5-fold dilution series and the 1 : 150 and 1 :3750 dilutions were used for the summary area under the curve (AUC) calculations shown on the heatmap of FIG. 36. Compared to wildtype (G34, corresponding to construct FLV.6029), soluble NS1 monomer encoding the above KO mutations (G33, corresponding to construct FLV.6028) elicited antibodies with lower cross-reactivity against flavivirus NS1 proteins. However, the KO mutations do not preclude cross-reactive responses, as exemplified by G31, which corresponds to membrane-anchored WNV NS1 monomer fused to EDN (FLV.6026).

[0556] EDN tags are critical for immunogenicity of monomeric, membrane-anchored NS1 designs. Specifically, G30 (MPMV.HIV as EDN tag, construct FLV.6025) and G31 (EABRsh as EDN tag, construct FLV.6026), but not G32 (no EDN, construct FLV.6027) elicited binding antibodies against WNV NS 1. Additionally, G31 elicited stronger and more cross-reactive antibody responses compared to G30. Thus, EDN tags may differ in their ability to promote strong and / or cross-reactive antibody responses.

[0557] EDN tags are also important for immunogenicity of homodimeric, membrane-anchored WNV NS1 designs, as demonstrated by comparing G26, G27 (both without EDN) to G22, G23, G20, G21 (all have EDN tag). Two different TM domains (EBV gp220 or measles HA) and two different EDN tags (EABRsh or MPMV.HIV) were tested. TM domain and EDN tag identity had minimal effects on immunogenicity in this context.

[0558] The immunogenicity of heterodimeric antigens depended on antigen composition. Specifically, ZIKV-DENV1 heterodimers (G25, G29; constructs FLV.6020, FLV.6024), but not WNV-JEV heterodimers (G24, G28; constructs FLV.6019, FLV.6023) elicited NS1 bindingAttorney Docket No.: VCCN-016 / 01WO 347597-2099 antibodies. Notably, ZIKV-DENV1 heterodimers elicited antibodies that were cross-reactive with NS1 from flaviviruses (DENV2, DENV3, DENV4) not included in the antigen composition.Example 8: Membrane-anchored NS1 antigens fused to EDN elicit antibodies with similar epitope specificities as mAbs with protective efficacy

[0559] Antibodies that target epitopes on the cell-surface form of NS1 are associated with in vivo protective efficacy. A competition ELISA as depicted in FIG. 37 was performed with a panel of mAbs against WNV NS1 with low (WNV-97), moderate (WNV-99), and high (WNV- 96, WNV-98, 22NS1) binding avidities to cell-surface NS1. As shown FIGS. 38A-38B, NS1 binding of serum antibodies from mice immunized with the cell-anchored WNV NS1 monomeric and homodimeric antigens fused to EDN were selectively competed by mAbs with high avidity to cell-surface forms of NS1, suggesting that these antigen designs can elicit antibodies with epitope specificities correlated with protective efficacy.Example 9: Additional animal studies

[0560] Mice and rabbits are used in immunization and in vivo toxicology studies for testing vaccine candidates. As shown in FIG. 7, vaccine candidates in an mRNA formulation can be administered intramuscularly to mice and rabbits, in a single dose or in multiple doses, with vehicle control such as, phosphate-buffered saline (PBS), administered to control animals. FIG. 7 also provides an example of a vaccination schedule. After immunization, plasma or serum is isolated from each animal and used in neutralization and binding assays to determine (1) the vaccine candidates’ ability to prevent or enhance flavivirus infection in vitro, (2) the vaccine candidates’ ability to induce antibodies that target relevant proteins and epitopes, and (3) presence of inflammatory cytokines such as IL-6 and TNF-alpha. The animals’ weight will also be monitored during the course of the studies.

[0561] Vaccine candidates are further tested in non-human primates (NHP), as shown in FIG. 8, through NHP challenge (top) and durability (bottom) experiments. In the NHP challenge experiment, half the animals receive vaccine and the other half receive placebo. Half of the animals within each vaccine or placebo group are challenged with Zika virus and the other half with dengue virus. At the predicted time of peak vaccine-induced immunity, the NHPs are subsequently challenged with the heterologous virus (i.e., Zika followed by dengue challenge and vice versa). NHP durability experiments are conducted such that a single dose or two doses are administered and antisera is tested at timed intervals for binding and neutralizing antibodies.Attorney Docket No.: VCCN-016 / 01WO 347597-2099Plasma will also be collected for viral load measurements by quantitative reverse transcriptase PCR.

[0562] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0563] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0564] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

Attorney Docket No.: VCCN-016 / 01WO 347597-2099CLAIMSI / We claim:

1. A modified flavivirus E polypeptide comprising at least one modification relative to a naturally occurring E polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

2. The modified flavivirus E polypeptide of claim 1, wherein the at least one improved characteristic comprises increased stability, increased immunogenicity, improved breadth of immunogenicity, increased secretion as enveloped virus-like particles (eVLPs), improved ability to form a lattice, improved ability to make mosaics, improved ability to make mosaic lattices, improved expression, or decreased potential to induce antibody-dependent enhancement (ADE).

3. The modified flavivirus E polypeptide of claim 1 or 2, wherein the modified polypeptide exhibits increased stability in an acidic environment or at physiological temperatures.

4. The modified flavivirus E polypeptide of any one of claims 1-3, wherein the at least one modification is ablation of an immunodominant ADE-inducing epitope within the E fusion loop and whereby the modified polypeptide exhibits decreased potential to induce ADE.

5. The modified flavivirus E polypeptide of any one of claims 1-4, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

6. The modified flavivirus E polypeptide of any one of claims 1-5, wherein the at least one modification comprises an amino acid deletion.

7. The modified flavivirus E polypeptide of any one of claims 1-6, wherein the at least one modification comprises an amino acid substitution.

8. The modified flavivirus E polypeptide of any one of claims 1-7, wherein the at least one modification comprises an amino acid insertion.

9. The modified flavivirus E polypeptide of claim 8, wherein the amino acid insertion comprises insertion of an endoplasmic reticulum (ER) signal sequence, a portion of a flavivirus nonstructural (NS) protein, an ER export sequence, an ESCRT recruiting domain (ERD), anAttorney Docket No.: VCCN-016 / 01WO 347597-2099ESCRT-independent eVLP inducing domain, a potential N-linked glycosylation site (PNGS) or a combination thereof.

10. The modified flavivirus E polypeptide of claim 9, wherein the amino acid insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5.

11. The modified flavivirus E polypeptide of claim 9 or 10, wherein the flavivirus nonstructural protein is NS2 or NS3.

12. The modified flavivirus E polypeptide of any one of claims 9-11, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD.

13. The modified flavivirus E polypeptide of any one of claims 9-12, the amino acid insertion comprises insertion of an ERD, optionally wherein the ERD comprises the sequence of any one of SEQ ID NOS: 350-387 or a sequence having at least 70% sequence identity thereto.

14. The modified flavivirus E polypeptide of claim 9, wherein the insertion comprises an ER export sequence and wherein the export sequence comprises the YTDIEM motif (SEQ ID NO: 144).

15. The modified flavivirus E polypeptide of any one of claims 1-14, wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), or Murray Valley Encephalitis virus (MVEV).

16. The modified flavivirus E polypeptide of any one of claims 1-15, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 1-17 or a truncated sequence thereof.

17. A modified NS1 polypeptide comprising at least one modification relative to a naturally occurring NS 1 polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic, wherein the characteristic is increased stability, increased immunogenicity, decrease in capacity to trigger endothelial barrier permeability, improved ability to form stable oligomers for display in various forms, or increased breadth of the resultant immune response.Attorney Docket No.: VCCN-016 / 01WO 347597-209918. The modified flavivirus NS1 polypeptide of claim 17, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

19. The modified flavivirus NS1 polypeptide of claim 17 or 18, wherein the at least one modification comprises an amino acid deletion.

20. The modified flavivirus NS1 polypeptide of any one of claims 17-19, wherein the at least one modification comprises an amino acid substitution.

21. The modified flavivirus NS1 polypeptide of claim 20, wherein the amino acid substitution comprises substitution of an amino acid at a position corresponding to position 115, 118, 119, or 207 of SEQ ID NO: 26.

22. The modified flavivirus NS1 polypeptide of claim 20 or 21, wherein the amino acid substitution comprises substitution of an amino acid at a position corresponding to position 115 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A).

23. The modified flavivirus NS1 polypeptide of any one of claims 20-22, wherein the amino acid substitution comprises:(a) substitution of an amino acid at a position corresponding to position 118 of SEQ ID NO: 26, wherein the amino acid substitution is Tryptophan (W) to Alanine (A);(b) substitution of an amino acid at a position corresponding to position 119 of SEQ ID NO: 26, wherein the amino acid substitution is Glycine (G) to Alanine (A);(c) substitution of an amino acid at a position corresponding to position 207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q); and / or(d) substitution of an amino acid at a position corresponding to position 207 of SEQ ID NO: 26, wherein the amino acid substitution is Asparagine (N) to Glutamine (Q).

24. The modified flavivirus NS1 polypeptide of claim 20, wherein the amino acid substitution comprises substitution of a wing domain of the NS1 polypeptide with a wing domain of a different flavivirus NS1 polypeptide.

25. The modified flavivirus NS1 polypeptide of claim 24, wherein the wing domain of a WNV NS1 polypeptide is substituted with a wing domain of ZIKV, JEV, or DENV NS1 polypeptide.Attorney Docket No.: VCCN-016 / 01WO 347597-209926. The modified flavivirus NS1 polypeptide of claim 24, wherein the wing domain of a different flavivirus NS1 polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 43-46, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

27. The modified flavivirus NS1 polypeptide of any one of claims 18-26 wherein the at least one modification comprises an amino acid insertion.

28. The modified flavivirus NS1 polypeptide of claim 27, wherein the amino acid insertion comprises insertion of an ER signal sequence, a portion of a flavivirus NS protein, an ER export sequence, an ESCRT recruiting domain (ERD), an ESCRT -independent eVLP inducing domain, a transmembrane domain, or a combination thereof.

29. The modified flavivirus NS1 polypeptide of claim 28, wherein the insertion comprises insertion of a portion of a flavivirus nonstructural protein and wherein the flavivirus nonstructural protein is NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5.

30. The modified flavivirus NS1 polypeptide of claim 28 or 29, wherein the flavivirus nonstructural protein is NS2 or NS3.

31. The modified flavivirus NS1 polypeptide of any one of claims 28-30, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain or an ERD.

32. The modified flavivirus NS1 polypeptide of claim 31, wherein the portion of the flavivirus nonstructural protein comprises a transmembrane domain.

33. The modified flavivirus NS1 polypeptide of claim 32, wherein the transmembrane domain comprises the amino acid sequence of the transmembrane domain of any one of SEQ ID NOs: 20 to 34 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

34. The modified flavivirus NS1 polypeptide of any one of claims 18-33, wherein the insertion comprises an ER export sequence and wherein the export sequence comprises the YTDIEM motif (SEQ ID NO: 144).

35. The modified flavivirus NS1 polypeptide of any one of claims 18-34, wherein the insertion comprises a transmembrane domain.Attorney Docket No.: VCCN-016 / 01WO 347597-209936. The modified flavivirus NS1 polypeptide of claim 35, wherein the transmembrane domain is 20, 21, 22, 23, or 24 amino acids in length.

37. The modified flavivirus NS1 polypeptide of claim 36, wherein the transmembrane domain is 21 or 22 amino acids in length.

38. The modified flavivirus NS1 polypeptide of any one of claims 35-37 wherein the transmembrane domain is derived from a bacterial or viral protein.

39. The modified flavivirus NS1 polypeptide of claim 38, wherein the bacterial or viral protein is selected from the group consisting of Epstein-Barr Virus (EBV) gp220 protein, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 M protein, Bovine viral diarrhea virus (BVDV) E2 protein, SARS-CoV-2 E protein, E. coli PmrR protein, Salmonella enterica (S. enterica) MgtU protein, Mycobacterium KdpF protein, Herpes Simplex Virus-2 (HSV-2) gH protein, Human Cytomegalovirus (HCMV) gH protein, Influenza A (FluA) M2 protein, FluA hemagglutinin protein, Measles virus hemagglutinin protein, Sendai virus (SeV) F protein, Vesicular Stomatitis Virus (VSV) G protein, Simian Vacuolating Virus 40 (SV40) Agnoprotein, Monkeypox virus (Mpox) A35R protein, and Sindbis virus (SINV) El protein.

40. The modified flavivirus NS1 polypeptide of any one of claims 35-37, wherein the transmembrane domain is derived from a platelet-derived growth factor receptor (PDGFR).

41. The modified flavivirus NS1 polypeptide of claim 40, wherein the transmembrane domain is derived from a human PDGFR (hPDGFR) or common sole PDGFR (caPDGFR)42. The modified flavivirus NS1 polypeptide of any one of claims 35-41 wherein the transmembrane domain comprises the amino acid sequence of any one of SEQ ID NOS: 110- 134, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

43. The modified flavivirus NS1 polypeptide of any one of claims 35-42, wherein the transmembrane domain is inserted at the N- or C-terminus of the NS1 polypeptide.

44. The modified flavivirus NS1 polypeptide of any one of claims 35-43 wherein the insertion comprises (a) a transmembrane (TM) domain and (b) an ERD or an ESCRT- independent eVLP inducing domain.Attorney Docket No.: VCCN-016 / 01WO 347597-209945. The modified flavivirus NS1 polypeptide of claim 44, wherein the insertions are made at the C-terminus of the NS1 polypeptide and comprise, from N-terminus to C-terminus:(a) a TM domain and an ERD; or(b) a TM domain and an ESCRT-independent eVLP inducing domain.

46. The modified flavivirus NS1 polypeptide of any one of claims 35-43, wherein the insertion comprises (a) a TM domain, (b) an endocytosis prevention motif (EPM), and (c) an ERD or an ESCRT-independent eVLP inducing domain.

47. The modified flavivirus NS1 polypeptide of claim 46, wherein the insertions are made at the C-terminus of the NS1 polypeptide and comprise, from N-terminus to C-terminus:(a) a transmembrane (TM) domain, an EPM, and an ERD; or(b) a transmembrane (TM) domain, an EPM, and an ESCRT-independent eVLP inducing domain.

48. The modified flavivirus NS1 polypeptide of any one of claims 18-47, comprising an amino acid insertion comprising an ERD, wherein the ERD comprises the sequence of any one of SEQ ID NOS: 350-387 or a sequence having at least 70% sequence identity thereto.

49. The modified flavivirus NS1 polypeptide of any one of claims 17-47 wherein the at least one modification is relative to a naturally occurring NS1 protein sequence from a flavivirus of the Orthofl avirvir us genus, optionally wherein the flavivirus is West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

50. The modified flavivirus NS1 polypeptide of any one of claims 17-49, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 20 to 34 or a truncated sequence thereof.

51. The modified flavivirus NS1 polypeptide of any one of claims 17-50, wherein the amino acid sequence of the modified polypeptide comprises the sequence of any one of SEQ ID NOS: 35 to 42, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.Attorney Docket No.: VCCN-016 / 01WO 347597-209952. The modified flavivirus NS1 polypeptide of any one of claims 17-51, wherein the amino acid sequence of the modified polypeptide comprises the sequence of any one of SEQ ID NOS: 278-311 and 324-336, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

53. A plurality of modified flavivirus NS1 polypeptides selected from any one of claims 17- 50, wherein the plurality of NS1 polypeptides are fused into a single polypeptide chain, and wherein the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides with identical or non-identical sequences.

54. The plurality of modified flavivirus NS1 polypeptides of claim 53, wherein the at least two modified NS1 polypeptides comprise a NSl polypeptide derived from a first flavivirus and a NS1 polypeptide derived from a second flavivirus.

55. The plurality of modified flavivirus NS1 polypeptides of claim 54, wherein the first flavivirus and the second flavivirus are the same.

56. The plurality of modified flavivirus NS1 polypeptides of claim 54, wherein the first flavivirus and the second flavivirus are different.

57. The plurality of modified flavivirus NS1 polypeptides of any one of claims 54-56, wherein the first flavivirus and the second flavivirus are selected independently from the group consisting of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

58. The plurality of modified flavivirus NS1 polypeptides of claim 57, wherein:(a) the first flavivirus is WNV and the second flavivirus is ZIKV;(b) the first flavivirus is WNV and the second flavivirus is JEV;(c) the first flavivirus is WNV and the second flavivirus is DENV;(d) the first flavivirus is ZIKV and the second flavivirus is JEV;(e) the first flavivirus is ZIKV and the second flavivirus is DENV; or(f) the first flavivirus is JEV and the second flavivirus is DENV;Attorney Docket No.: VCCN-016 / 01WO 347597-209959. The plurality of modified flavivirus NS1 polypeptides of any one of claims 53-58, comprising at least three modified NS1 polypeptides with non-identical sequences, wherein the at least three NS1 polypeptides comprise a NS1 polypeptide derived from a first flavivirus, a NS1 polypeptide derived from a second flavivirus, and a NS1 polypeptide derived from a third flavivirus.

60. The plurality of modified flavivirus NS1 polypeptides of 59, wherein the first flavivirus, the second, and the third flavivirus are selected independently from the group consisting of: West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickborne encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

61. The plurality of modified flavivirus NS1 polypeptides of claim 60, wherein:(a) the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is JEV;(b) the first flavivirus is WNV, the second flavivirus is ZIKV, and the third flavivirus is DENV;(c) the first flavivirus is WNV, the second flavivirus is JEV, and the third flavivirus is DENV; or(d) the first flavivirus is ZIKV, the second flavivirus is JEV, and the third flavivirus is DENV.

62. The plurality of modified flavivirus NS1 polypeptides of any one of claims 53-61, wherein the plurality of NS1 polypeptides comprises at least two modified NS1 polypeptides, wherein the sequences of the at least two modified NS1 polypeptides are not identical, thereby improving the breadth of immunogenicity.

63. The plurality of modified flavivirus NS1 polypeptides of any one of claims 53-62, comprising the amino acid sequence of any one of SEQ ID NOS: 286-295, or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto, or a sequence having at least 70% sequence identity thereto.

64. The plurality of modified flavivirus NS1 polypeptides of any one of claims 53-63, wherein the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice.Attorney Docket No.: VCCN-016 / 01WO 347597-209965. The plurality of modified flavivirus NS1 polypeptides of any one of claims 53-64, comprising two, three, or five modified NS 1 polypeptides selected from any one of claims 17- 52, wherein the sequences of the two, three, or five modified NS1 polypeptides are not identical, optionally wherein the modified NS1 polypeptides have orthogonal interfaces.

66. The plurality of modified flavivirus NS1 polypeptides of any one of claims 53-65, wherein the plurality of polypeptides is capable of assembling into a stochastic mosaic, optionally comprising six, seven, or eight modified NS1 polypeptides with identical interfaces, wherein the sequences of the six, seven, or eight modified NS1 polypeptides are not identical.

67. A modified flavivirus prM polypeptide comprising at least one modification relative to a naturally occurring prM polypeptide, wherein the modified polypeptide exhibits at least one improved characteristic.

68. The modified flavivirus prM polypeptide of claim 67, wherein the characteristic is increased ability to stabilize the conformation of naturally occurring or modified flavivirus E, improved expression, improved secretion, a decrease in immature, conformationally heterogeneous virions, or a combination thereof.

69. The modified flavivirus prM polypeptide of claim 67 or 68, wherein the at least one modification comprises a mutation, a deletion, an insertion, and / or an amino acid substitution.

70. The modified flavivirus prM polypeptide of any one of claims 67-69, wherein the at least one modification comprises an amino acid deletion.

71. The modified flavivirus prM polypeptide of claim 70, wherein the amino acid deletion comprises deletion of the pr amino acid sequence or a portion thereof, optionally wherein the pr amino acid sequence comprises the sequence of any one of SEQ ID NOs: 70-77 or a sequence having at least 1, 2, 3, 4, 5 amino acid variations thereto or having at least 70% sequence identity thereto.

72. The modified flavivirus prM polypeptide of any one of claims 67-71, wherein the at least one modification comprises an amino acid substitution.

73. The modified flavivirus prM polypeptide of any one of claims 67-72, wherein the at least one modification comprises an amino acid insertion.Attorney Docket No.: VCCN-016 / 01WO 347597-209974. The modified flavivirus prM polypeptide of any one of claims 67-73, wherein the amino acid sequence of the modified polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NOs: 50-67 or a truncated sequence thereof.

75. The modified flavivirus E polypeptide of any one of claims 1-16, further fused directly or through a linker to a second flavivirus polypeptide.

76. The modified flavivirus E polypeptide of claim 75, wherein the second flavivirus polypeptide comprises a naturally occurring prM polypeptide or the modified flavivirus prM polypeptide of any one of claims 67-73, a naturally occurring NS1 or the modified flavivirus NS1 polypeptide of any one of claims 17-52, and / or the plurality ofNSl polypeptides of any one of claims 53-66.

77. The modified flavivirus E polypeptide of claim 76, wherein the second flavivirus polypeptide is fused directly or through a linker to the N- or C-terminus of the modified flavivirus E polypeptide.

78. The modified flavivirus E polypeptide of any one of claims 75-77, wherein the second flavivirus polypeptide comprises a naturally occurring prM or the modified flavivirus prM polypeptide of any one of claims 67-73; and wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide.

79. The modified flavivirus E polypeptide of any one of claims 75-77, wherein the second flavivirus polypeptide comprises a naturally occurring NS1 or the modified flavivirus NS1 polypeptide of any one of claims 17-52 or the plurality ofNSl polypeptides of any one of claims 53-66; and wherein the second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

80. The modified flavivirus E polypeptide of any one of claims 75-79 further fused directly or through a linker to a third flavivirus polypeptide.

81. The modified flavivirus E polypeptide of claim 80, wherein the third flavivirus polypeptide comprises a naturally occurring prM polypeptide or the modified flavivirus prM polypeptide of any one of claims 67-73, the modified flavivirus NS1 polypeptide of any one of claims 17-52, and / or the plurality ofNSl polypeptides of any one of claims 53-66.Attorney Docket No.: VCCN-016 / 01WO 347597-209982. The modified flavivirus E polypeptide of claim 81, wherein the second flavivirus polypeptide comprises a naturally occurring prM or the modified flavivirus prM polypeptide of any one of claims 67-73 and the third flavivirus polypeptide comprises a naturally occurring NS1 or the modified flavivirus NS1 polypeptide of any one of claims 17-52 or the plurality of NS1 polypeptides of any one of claims 53-66; and wherein the second flavivirus polypeptide is fused directly or through a linker to the N terminus of the modified flavivirus E polypeptide and the third second flavivirus polypeptide is fused directly or through a linker to the C terminus of the modified flavivirus E polypeptide.

83. The modified flavivirus E polypeptide of any one of claims 75-82, comprising a protease cleavage site.

84. The modified flavivirus E polypeptide of claim 79 or 82, comprising a protease cleavage site, wherein the protease cleavage site is located between the modified flavivirus E polypeptide and the modified flavivirus NS1 polypeptide or the plurality ofNSl polypeptides.

85. The modified flavivirus E polypeptide of claim 83 or 84, wherein the protease cleavage site is a naturally occurring cleavage site in a flavivirus, optionally wherein the cleavage site is a host cleavage site.

86. The modified flavivirus E polypeptide of any one of claims 75-85, wherein the modified flavivirus E polypeptide comprises the sequence of any one of SEQ ID NOS: 150-173, 176-183, 185-227, 229-270, 272-277, and 312-323 or a sequence having at least 70% sequence identity thereto.

87. A plurality of modified flavivirus E polypeptides selected from any one of claims 1-16 and 75-86, wherein the plurality of polypeptides are fused into a single chimeric polypeptide chain.

88. The plurality of modified flavivirus E polypeptides of claim 87, wherein the plurality of polypeptides is capable of assembling into a mosaic or mosaic lattice.

89. The plurality of modified flavivirus E polypeptides of claim 87 or 88, comprising two, three, or five modified E polypeptides selected from any one of claims 1-16 and 75-86, wherein the sequences of the comprising two, three, or five modified E polypeptides are not identical, optionally wherein the modified E polypeptides have orthogonal interfaces.Attorney Docket No.: VCCN-016 / 01WO 347597-209990. The plurality of modified flavivirus E polypeptides of any one of claims 87-89, wherein the plurality of polypeptides is capable of assembling into a stochastic mosaic, optionally comprising six, seven, or eight modified E polypeptides with identical interfaces, wherein the sequences of the six, seven, or eight modified E polypeptides are not identical.

91. A polynucleotide encoding the amino acid sequence of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90.

92. The polynucleotide of claim 91, wherein the polynucleotide comprises DNA.

93. The polynucleotide of claim 91, wherein the polynucleotide comprises RNA.

94. The polynucleotide of claim 93, wherein the polynucleotide comprises mRNA.

95. The polynucleotide of claim 94, wherein the mRNA comprises modified nucleosides.

96. A vector comprising the polynucleotide of any one of claims 91-95 or a plurality of polynucleotides selected from any one of claims 91-95.

97. The vector of claim 96, wherein the vector is a viral vector.

98. The vector of claim 97, wherein the viral vector is an adeno-associated virus (AAV) vector, adenovirus vector, modified vaccinia virus Ankara (MV A) vector, poxvirus vector, or Vesicular stomatitis virus (VSV) vector.

99. The vector of claim 98, wherein the adenovirus vector is Ad26, Ad34, Ad35, Ad48, or Ad5HVR48.

100. The vector of claim 96, wherein the vector is a non-viral vector.

101. The vector of claim 100, wherein the non-viral vector is a plasmid.

102. The vector of claim 100, wherein the non-viral vector is a lipid nanoparticle (LNP).

103. A cell comprising the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modifiedAttorney Docket No.: VCCN-016 / 01WO 347597-2099 flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90.

104. A cell displaying on its cell surface all or a portion thereof of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90.

105. An enveloped virus-like particle (eVLP) comprising the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90.

106. An enveloped virus-like particle (eVLP) displaying all or a portion thereof of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90.

107. A vaccine composition comprising the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96-102, the cell of claim 103 or 104, or the eVLP of claim 105 or 106.

108. The vaccine composition of claim 107, further comprising one or more adjuvants.

109. The vaccine composition of claim 107 or 108, further comprising one or more pharmaceutically acceptable carrier, excipient, or diluent.

110. A kit comprising the modified flavivirus E polypeptide of any one of claims 1-16 and 75- 86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modifiedAttorney Docket No.: VCCN-016 / 01WO 347597-2099 flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96-102, the cell of claim 103 or 104, the eVLP of claim 105 or 106, or the vaccine composition of any one of claims 107-109; and instructions for use.

111. A method of preventing a flavivirus infection or a symptom thereof in a subject having, or at risk of having, a flavivirus infection, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67- 74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96-102, the cell of claim 103 or 104, the eVLP of claim 105 or 106, or the vaccine composition of any one of claims 107-109.

112. The method of preventing a flavivirus infection of claim 111, wherein the subject has, or is at risk of having, an infection of West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZV), Powassan virus (POWV), Langat virus (LGTV), Usutu virus (USUV), tickbome encephalitis virus (TBEV), Spondweni virus (SPOV), St. Louis encephalitis virus (SLEV), or Murray Valley Encephalitis virus (MVEV).

113. The method of preventing a flavivirus infection of claim 111 or 112, wherein the subject is a mammal or a bird, optionally wherein the subject is pregnant.

114. The method of preventing a flavivirus infection of claim 113, wherein the subject is a mammal.

115. The method of preventing a flavivirus infection of claim 114, wherein the subject is a human, optionally wherein the human subject is pregnant.

116. The method of preventing a flavivirus infection of claim 113, wherein the subject is a bird.Attomey Docket No.: VCCN-016 / 01WO 347597-2099117. A method of treating a flavivirus infection or a symptom thereof in a subject having a flavivirus infection, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96-102, the cell of claim 103 or 104, the eVLP of claim 105 or 106, or the vaccine composition of any one of claims 107-109.

118. A method of inducing anti-flavivirus antibodies in a subject, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of claims 1- 16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96- 102, the cell of claim 103 or 104, the eVLP of claim 105 or 106, or the vaccine composition of any one of claims 107-109.

119. The method of claim 118, wherein the anti-flavivirus antibodies target E protein, M protein, and / or NS1 protein, optionally wherein the anti-flavivirus antibodies target proteins of at least two flaviviruses.

120. A method for inducing a flavivirus-specific immune response, comprising administering to the subject an effective amount of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96- 102, the cell of claim 103 or 104, the eVLP of claim 105 or 106, or the vaccine composition of any one of claims 107-109, optionally wherein the immune response provides immune protection for at least two flaviviruses.

121. The modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67-Attorney Docket No.: VCCN-016 / 01WO 347597-209974, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96-102, the cell of claim 103 or 104, the eVLP of claim 105 or 106, or the vaccine composition of any one of claims 107-109 for use in the treatment of a flavivirus infection or a symptom thereof.

122. Use of the modified flavivirus E polypeptide of any one of claims 1-16 and 75-86, the modified NS1 polypeptide of any one of claims 17-52, the plurality of modified flavivirus NS1 polypeptide of any one of claims 53-66, the modified prM polypeptide of any one of claims 67- 74, or the plurality of modified flavivirus E polypeptides of any one of claims 87-90, the polynucleotide of any one of claims 91-95, the vector of any one of claims 96-102, the cell of claim 103 or 104, the eVLP of claim 105 or 106 or the vaccine composition of any one of claims 107-109 in the manufacture of a medicament for the treatment of a flavivirus infection or a symptom thereof.

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